Patent Application
20250034626
This application contains a sequence listing, submitted electronically in ASCII format under the filename Sequence_Listing.txt, which is incorporated by reference herein in its entirety. The ASCII copy of the sequence listing was created on Oct. 16, 2023, and is 21,908 bytes in size.
This invention relates to a method of detecting the status of epigenetic modification of a target polynucleotide sequence in a given nucleic acid analyte in particular a method for testing for the presence of a large number of methylated markers, including those used in the identification of cancer, infectious disease and transplant organ rejection. It is also useful for companion diagnostic testing in which a panel of markers must be identified reliably and at low cost.
In the chemical sciences, methylation denotes the addition of a methyl group to a substrate or the substitution of an atom or group by a methyl group. Methylation is a form of alkylation with specifically a methyl group, rather than a larger carbon chain, replacing a hydrogen atom. These terms are commonly used in chemistry, biochemistry, soil science, and the biological sciences.
In biological systems, methylation is catalysed by enzymes: such methylation can be involved in modification of heavy metals, regulation of gene expression, regulation of protein function, and RNA metabolism. Methylation of heavy metals can also occur outside of biological systems. Chemical methylation of tissue samples is also one method for reducing certain histological staining artefacts.
Aberrant DNA methylation profiles have been associated many different complex disease states. In oncology, hypermethylation of tumour suppressor genes in serum DNA can be used as a diagnostic marker for small-cell lung cancer. In patients with immune disease, such as diabetes, rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE), abnormal DNA methylation of cells of the immune system is found. Differential DNA methylation of peripheral blood leukocytes (PBL) in repetitive elements ALU, LINE-1 and Satellite 2 (measures for global DNA methylation), have been found to be associated with ischemic heart disease.
DNA methylation in vertebrates typically occurs at CpG sites (Cytosine-phosphate-guanine sites; that is, where a cytosine is directly followed by a guanine in the DNA sequence); this methylation results in the conversion of the cytosine to 5-methylcytosine. The formation of Me-CpG is catalysed by the enzyme DNA methyltransferase. The bulk of mammalian DNA has about 40% of CpG sites methylated but there are certain areas, known as CpG islands which are GC rich (made up of about 65% CG residues) where none are methylated. These are associated with the promoters of 56% of mammalian genes, including all ubiquitously expressed genes. 1-2% of the human genome is CpG clusters and there is an inverse relationship between CpG methylation and transcriptional activity.
DNA methylation involves the addition of a methyl group to the 5 position of cytosine pyrimidine ring or the 6 nitrogen of the adenine purine ring. This modification can be inherited through cell division. DNA methylation is typically removed during zygote formation and re-established through successive cell divisions during development. DNA methylation is a crucial part of normal organism development and cellular differentiation in higher organisms. DNA methylation stably alters the gene expression pattern in cells such that cells can “remember where they have been”; in other words, cells programmed to be pancreatic islets during embryonic development remain pancreatic islets throughout the life of the organisms without continuing signals telling them that they need to remain islets. In addition, DNA methylation suppresses the expression of viral genes and other deleterious elements which have been incorporated into the genome of the host over time. DNA methylation also forms the basis of chromatin structure, which enables cells to form the myriad characteristics necessary for multicellular life from a single immutable sequence of DNA. DNA methylation also plays a crucial role in the development of nearly all types of cancer. Bisulfite sequencing is the use of bisulfite treatment of DNA to determine its pattern of methylation. DNA methylation was the first discovered epigenetic mark, and remains the most studied. It is also implicated in repression of transcriptional activity.
Among a number of mRNA modifications, N6-methyladenosine (m6A) modification is the most common type in eukaryotes and nuclear-replicating viruses. m6A has a significant role in numerous cancer types, including leukaemia, brain tumours, liver cancer, breast cancer and lung cancer.
Whilst 5-methylcytosine (5mC) is the most studied epigenetic modification, 5mC is oxidised to 5-hydroxymethylcytosine (5hmC) with the catalysis of TET (ten-eleven translocation) enzymes. Studies have shown that the distribution of 5hmC is tissue-specific, and there are differences in the distribution of 5hmC in different organs and tissues. The decreased expression of 5hmC in malignant tissue has been shown consistently in a wide range of different cancers, including melanoma. By evaluating a total of 15 pairs of normal and carcinoma samples in human breast tissue, the studies have shown that the levels of 5hmC were dramatically reduced in the cancer group compared with the healthy breast tissues.
Treatment of DNA with bisulfite converts cytosine residues to uracil, but leaves 5-methylcytosine residues unaffected. Thus, bisulfite treatment introduces specific changes in the DNA sequence that depend on the methylation status of individual cytosine residues, yielding single-nucleotide resolution information about the methylation status of a segment of DNA. Various analyses can be performed on the altered sequence to retrieve this information. The objective analysis is therefore reduced to differentiating between single nucleotide polymorphisms (cytosines and thymines) resulting from bisulfite conversion.
5hmC converts to 5mC upon bisulfite treatment, which then reads as a C when sequenced, and thus cannot distinguish between 5hmC and 5mC. The output from bisulfite sequencing can no longer be defined as solely DNA methylation, as it is the composite of 5mC and 5hmC. The development of TET-assisted oxidative bisulfite sequencing is now able to distinguish between the two modifications at single base resolution.
5hmC can be detected using TET-assisted bisulfite sequencing (TAB-seq). Fragmented DNA is enzymatically modified using sequential T4 Phage R-glucosyltransferase (T4-BGT) and then Ten-eleven translocation (TET) dioxygenase treatments before the addition of sodium bisulfite. T4-BGT glucosylates 5hmC to form beta-glucosyl-5-hydroxymethylcytosine (5ghmC) and TET is then used to oxidize 5mC to 5caC. Only 5ghmC is protected from subsequent deamination by sodium bisulfite and this enables 5hmC to be distinguished from 5mC by sequencing.
Oxidative bisulfite sequencing (oxBS) provides another method to distinguish between 5mC and 5hmC. The oxidation reagent potassium perruthenate converts 5hmC to 5-formylcytosine (5fC) and subsequent sodium bisulfite treatment deaminates 5fC to uracil. 5mC remains unchanged and can therefore be identified using this method.
APOBEC-coupled epigenetic sequencing (ACE-seq) excludes bisulfite conversion altogether and relies on enzymatic conversion to detect 5hmC. With this method, T4-BGT glucosylates 5hmC to 5ghmC and protects it from deamination by Apolipoprotein B mRNA editing enzyme subunit 3A (APOBEC3A). Cytosine and 5mC are deaminated by APOBEC3A and sequenced as thymine.
TET-assisted 5-methylcytosine sequencing (TAmC-seq) enriches for 5mC loci and utilizes two sequential enzymatic reactions followed by an affinity pull-down. Fragmented DNA is treated with T4-BGT which protects 5hmC by glucosylation. The enzyme mTET1 is then used to oxidize 5mC to 5hmC, and T4-BGT labels the newly formed 5hmC using a modified glucose moiety (6-N3-glucose). Click chemistry is used to introduce a biotin tag which enables enrichment of 5mC-containing DNA fragments for detection and genome wide profiling.
Methods of methylome analysis are broadly divided into 3 groups: restriction enzyme based, chromatin immunoprecipitation based (ChIP) or affinity based and bisulfite conversion (gene based). Restriction enzyme based methods are methylation-sensitive restriction enzymes for small/large scale DNA methylation analysis by combining the use of methylation-sensitive restriction enzymes experimental approaches (RLGS, DMH etc.) for global methylation analysis, applied to any genome without knowing the DNA sequence. However, large amounts of genomic DNA are required, making the method unsuitable for the analysis of samples when small amount of DNA is recovered. On the other hand, ChIP based methods are useful for the identification of differential methylated regions in tumours through the precipitation of a protein antigen out of a solution by using an antibody directed against the protein. These methods are protein based, applied extensively in cancer research.
Affinity enrichment is a technique that is often used to isolate methylated DNA from the rest of the DNA population. This is usually accomplished by antibody immunoprecipitation methods or with methyl-CpG binding domain (MBD) proteins.
Methylated DNA immunoprecipitation (MeDIP) is an antibody immunoprecipitation method that utilises a 5-methylcytidine antibody to specifically recognise methylated cytosines. The MeDIP kit requires the input DNA sample to be single-stranded in order for the 5-methylcytidine (5-mC) antibody to bind.
Another method for the enrichment of methylated DNA fragments uses recombinant methyl-binding protein MBD2b, or the MBD2b/MBD3L1 complex. One advantage of a methyl-CpG binding protein enrichment strategy is the input DNA sample does not need to be denatured; the protein can recognise methylated DNA in its native double-strand form. Another advantage is that the MBD protein binds only to DNA methylated in a CpG context to ensure the enrichment of methylated-CpG DNA, making this technique ideal for studying CpG islands.
The polymerase chain reaction (PCR) is a well-known and powerful technique for amplifying methylated DNA or RNA present in laboratory and diagnostic samples to a point where they can be reliably detected and/or quantified. However, when applied for the purposes of investigating analyte samples containing low-levels of such molecules, it suffers from a number of limitations. First, whilst the technique can detect as little as a single target molecule, it is prone to generating false positive results due to unwanted amplification of other nucleic acid sequences present in the sample. This makes the choice of oligonucleotide primers used to initiate the reaction key; which in turn makes designing primers with the required level of specificity relatively complex. As a consequence, many PCR-based tests available on the market today have limited specificity.
A second drawback is that multiplexing of PCR-based methods is in practice limited to at most tens of target sequences (frequently no more than 10) with the avoidance of primer-primer interactions resulting in the need for relatively narrow operational windows.
Another issue is that, because the PCR reaction cycles in an exponential fashion, quantification of the target is difficult; small variations in the efficiency of the reaction having a huge impact on the amount of detectable material generated. Even with appropriate controls and calibrations in place, quantification is thus typically limited to an accuracy within a factor of around 3.
Finally, mutations in the region targeted for investigation by PCR amplification methods can have unwanted side effects. For example, there have been instances where FDA-approved tests have had to be withdrawn because the target organism underwent mutation in the genetic region targeted by the test primers resulting in large numbers of false negatives. Conversely, if a specific single nucleotide polymorphism (SNP) is targeted for amplification the PCR method will often give a false positive when the wild-type variant is present. Avoiding this requires very careful primer design and further limits the efficacy of multiplexing. This is particularly relevant when searching for panels of SNPs as is a common requirement in cancer testing/screening or companion diagnostics.
US2006/110765 A1 (Wang et al) teaches enzymatic cleavage at a mismatch, which is typically an inefficient and not a highly specific reaction. Moreover, off target hybridisation of the probes as disclosed in Wang et al to similar sequences in a sample would result in false positive results because of the use of cleavage at mismatches. It would also not be possible to distinguish between two different genetic variants in the same or near-neighbouring positions as they would all result in cleavage and amplification of the same probe. The teachings of Wang et al would therefore result in low sensitivity and specificity of the reaction scheme. In contrast, the technical effect of the method as disclosed by the present invention provides a fast, efficient method with high specificity to dsDNA, which can be effectively blocked by a mismatch. In addition, the method of the present invention is extremely specific to the targeted genetic variant, enabling discrimination between different variants in the same or near-neighbouring positions.
US2009/239283 A1 (Liu et al) teaches the use of non-extendable 3′ ends which are removed by pyrophosphorolysis, necessitating the genetic engineering of custom polymerases capable of removing the 3′ blocking modification. In contrast the present invention utilises the natural pyrophosphorolysis activity inherent in existing polymerases and does not use 3′ blocking modifications. The method as disclosed in Liu et al also relies on the removal of only the terminal base from a fraction of the probe to enable subsequent amplification, and is limited to this embodiment by the use of 3′ blocking modification. In contrast, the methods disclosed in the present invention enables embodiments, in which progressive removal of multiple bases from the probe is required to set off the reaction, making it substantially more robust to transient off-target annealing either to the background DNA or to other probes, which can result in the unwanted removal of the terminal base.
We have now developed a new method for detecting the status of epigenetic modification of a target polynucleotide sequence in a given nucleic acid analyte, a method which overcomes many of the prior limitations. In doing so, it harnesses the double-strand specificity of pyrophosphorolysis; a reaction which will not proceed efficiently with single-stranded oligonucleotide substrates or double-stranded substrates which include blocking groups or nucleotide mismatches. Thus, according to the present invention, there is provided a method of detecting the epigenetic modification status of a target polynucleotide sequence in a given nucleic acid analyte characterised by the steps of:
The analytes to which the method of the invention can be applied are those nucleic acids, such as naturally-occurring or synthetic DNA or RNA molecules, which include the target polynucleotide sequence(s) being sought. In one embodiment, the analyte will typically be present in an aqueous solution containing it and other biological material and in one embodiment the analyte will be present along with other background nucleic acid molecules which are not of interest for the purposes of the test. In some embodiments, the analyte will be present in low amounts relative to these other nucleic acid components. Preferably, for example where the analyte is derived from a biological specimen containing cellular material, prior to performing step (a) of the method some or all of these other nucleic acids and extraneous biological material will have been removed using sample-preparation techniques such as filtration, centrifuging, chromatography or electrophoresis. Suitably, the analyte is derived from a biological sample taken from a mammalian subject (especially a human patient) such as blood, plasma, sputum, urine, skin or a biopsy. In one embodiment, the biological sample will be subjected to lysis in order that the analyte is released by disrupting any cells present. In other embodiments, the analyte may already be present in free form within the sample itself; for example cell-free DNA circulating in blood or plasma.
It will be obvious to one skilled in the art that the present invention may be extended towards the detection of any epigenetic modification and is not limited to the detection of methylation status of target polynucleotide sequences. For example, the present invention could equally be adapted for the detection of other epigenetic modifications including hydroxymethylation—for example the hydroxylated form of 5mC (5-hmC). This recently appreciated form of epigenetic modification is an important epigenetic marker which influences gene expression and is distinct from CpG methylation. Other epigenetic modifications appear on RNA such as methyl adenosine and can be detected by methods of the invention.
In step b, the partially double-stranded first intermediate product is pyrophosphorolysed with a pyrophosphorolysing enzyme in the 3′-5′ direction from the 3′ end of A0 to create a partially digested strand A1, the analyte and the undigested A0 molecule which did not anneal to a target in step a.
In step d, A2 is primed with at least one single-stranded primer oligonucleotide and multiple copies of A2, or a region of A2 are created.
In step d, A2 is primed with at least one single-stranded primer oligonucleotide and multiple copies of A2, or a region of A2 are created.
In step d, A2 is primed with at least one single-stranded primer oligonucleotide and multiple copies of A2, or a region of A2 are created.
According to the present invention, there is provided a method of detecting the epigenetic modification status of a target polynucleotide sequence in a given nucleic acid analyte characterised by the steps of:
In one embodiment, the 3′ end of A0 is perfectly complementary to the target polynucleotide sequence.
In one embodiment, the ligase is substantially lacking in single-strand ligation activity.
In one embodiment, (a) comprises chemically or enzymatically converting the unmethylated cytosine bases in the target polynucleotide sequence.
Thus in some embodiments, the method of detecting the epigenetic modification status of a target polynucleotide sequence in a given nucleic acid analyte comprises the steps of:
In one embodiment, unmodified cytosine bases are converted to uracil by a methyltransferase enzyme.
In one embodiment, this enzyme is M.Sssl
In one embodiment, unmodified cytosine bases are converted to uracil by a deaminase enzyme.
An enzymatic methyl-seq workflow relies on the ability of APOBEC to deaminate cytosines to uracils. APOBEC also deaminates 5mC and 5hmC, making it impossible to differentiate between cytosine and its modified forms. In order to detect 5mC and 5hmC, this method also utilizes TET2 and an Oxidation Enhancer, which enzymatically modifies 5mC and 5hmC to forms that are not substrates for APOBEC. The TET2 enzyme converts 5mC to 5caC and the Oxidation Enhancer converts 5hmC to 5ghmC. Ultimately, cytosines are sequenced as thymines and 5mC and 5hmC are sequenced as cytosines, thereby protecting the integrity of the original 5mC and 5hmC sequence information.
In some embodiments, the converted polynucleotide target is introduced to a restriction endonuclease prior to or during step (b).
In some embodiments, the recognition sequence of the restriction endonuclease is created by the conversion performed in step (a).
In an alternative embodiment, the recognition sequence of the restriction endonuclease is removed by the conversion performed in step (a) and A0 is prevented from undergoing pyrophosphorolysis through chemical modification at or close to its 3′ end, or through a 3′ mismatch against the target sequence, and this modification or mismatch is removed through cleavage of A0 by the restriction endonuclease prior to pyrophosphorolysis.
In one embodiment, (a) comprises introducing the nucleic acid analyte to an epigenetic modification-sensitive or epigenetic modification-dependent restriction endonuclease.
Thus in some embodiments, the method of detecting the epigenetic modification status of a target polynucleotide sequence in a given nucleic acid analyte comprises the steps of:
In one embodiment, the epigenetic modification-sensitive or epigenetic modification-dependent restriction endonuclease is McrBC.
In one embodiment, the epigenetic modification-sensitive or epigenetic modification-dependent restriction endonuclease is a member of the MspJI family.
In one embodiment, the endonuclease is AspBHI.
In one embodiment, the endonuclease is FspEl.
In one embodiment, the endonuclease is LpnPI,
In one embodiment, the epigenetic modification-sensitive or epigenetic modification-dependent restriction endonuclease is a member of the PvuRts1I/AbaS family.
In one embodiment, the epigenetic modification-sensitive or epigenetic modification-dependent restriction endonuclease is a Type IIM endonuclease.
In one embodiment, the endonuclease is DpnI.
In one embodiment, the endonuclease is BisI, In one embodiment, the epigenetic modification-sensitive or epigenetic modification-dependent restriction endonuclease is a Type IV endonuclease.
In one embodiment, the endonuclease is EcoKMcrBC.
In one embodiment, the endonuclease is SauUSI.
In one embodiment, the endonuclease is GmrSD.
In one embodiment, the epigenetic modification-sensitive or epigenetic modification-dependent restriction endonuclease is selected from the DpnII restriction endonuclease family.
In one embodiment, the endonuclease is DpnII.
In one embodiment, the endonuclease is DpnI.
In one embodiment, the epigenetic modification-sensitive or epigenetic modification-dependent restriction endonuclease is HpaI.
In one embodiment, the epigenetic modification-sensitive or epigenetic modification-dependent restriction endonuclease is HpaII.
In one embodiment, (a) comprises introducing the nucleic acid analyte to a methylation-sensitive or methylation-dependent restriction endonuclease.
Thus in some embodiments, the method of detecting epigenetic modification status of a target polynucleotide sequence in a given nucleic acid analyte comprises the steps of:
In some embodiments the method according to invention is where the epigenetic modification may be methylation. In further embodiments the epigenetic modification may be methylation at CpG islands or by hydroxymethylation at CpG islands.
In some embodiments the epigenetic modification may be methylation of adenine.
In some embodiments the method according to invention is where the epigenetic modification is methylation. In further embodiments the epigenetic modification is methylation at CpG islands or by hydroxymethylation at CpG islands.
In some embodiments the epigenetic modification is methylation of adenine in either RNA or DNA.
In some embodiments, the restriction endonuclease employed cleaves copies of the polynucleotide target sequence in which the target state is not present.
In some embodiments, the restriction endonuclease and the first reaction mixture are added at the same time.
In some embodiments, A0 is prevented from undergoing pyrophosphorolysis through chemical modification at or close to its 3′ end, or through a 3′ mismatch against the target sequence, and this modification or mismatch is removed through cleavage of A0 by the restriction endonuclease prior to pyrophosphorolysis. In some embodiments, this occurs during pyrophosphorolysis/during the pyrophosphorolysis step.
In some embodiments, wherein (a) comprises introducing the nucleic acid analyte to a methylation-sensitive or methylation-dependent restriction endonuclease, (a) further comprises selective amplification of the target polynucleotide sequence containing the methylation status of interest through methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) of methylated DNA.
In some embodiments, the method comprises the method according to any previous embodiment wherein the products of (a) undergo PCR prior to (b).
In some embodiments, the method comprises the method according to any previous embodiment wherein the population of methylated or unmethylated target sequence is reduced prior to step (a).
In some embodiments, the reduction is carried out using methylated DNA immunoprecipitation (MeDIP).
In some embodiments, the reduction is carried out using methyl-binding proteins, such as MBD2b or the MBD2b/MBD3L1 complex.
In some embodiments, the method comprises the method according to any previous embodiment wherein prior to step (c) the products of (b) are introduced to a second reaction mixture comprising at least one single-stranded primer oligonucleotide that is substantially complementary to a portion of A0, deoxyribonucleotide triphosphates (dNTPs) and an amplification enzyme.
In some embodiments, the first and second reaction mixtures are introduced concurrently.
In some embodiments, the dNTPs are hot start dNTPs.
Hot start dNTPs are dNTPs which are modified with a thermolabile protecting group at the 3′ terminus. The presence of this modification blocks DNA polymerase nucleotide incorporation until the nucleotide protecting group is removed using a heat activation step.
In some embodiments, the method comprises the method according to any previous embodiment wherein during step (b) the analyte anneals to the single-stranded probe oligonucleotide A0 to create a first intermediate product which is at least partially double-stranded and in which the 3′ end of A0 forms a double-stranded complex with the analyte target sequence and during step (b) the first intermediate product is pyrophosphorolysed in the 3′-5′ direction from the 3′ end of A0 to create partially digested strand A1 and the analyte.
In some embodiments, the method comprises the method according to any previous embodiment wherein the partially digested strand A1 is circularised through ligation of its 3′ and 5′ ends to create an oligonucleotide A2.
In some embodiments, the method comprises the method according to any previous embodiment wherein the first reaction mixture further comprises a ligation probe oligonucleotide C and that the partially digested strand A1 is ligated at the 3′ end to the 5′ end of C to create an oligonucleotide A2.
In some embodiments, the method comprises the method according to any previous embodiment wherein the oligonucleotide C further comprises a 3′ or internal modification protecting it from 3′-5′ exonuclease digestion.
In some embodiments, the ligation occurs:
In some embodiments, the first reaction mixture further comprises a 5′-3′ exonuclease and wherein the 5′ end of A0 is rendered resistant to 5′-3′ exonuclease digestion. In some embodiments, the method comprises the method according to any previous embodiment wherein the first reaction mixture further comprises a phosphatase or phosphohydrolase.
In some embodiments, the method comprises the method according to any previous embodiment wherein prior to or during step (c) the products of the previous step are treated with a pyrophosphatase.
In some embodiments, the method comprises the method according to any previous embodiment wherein that prior to or during step (c) the products of the previous step are treated with an exonuclease.
In some embodiments, the method comprises the method according to any previous embodiment wherein the first or second reaction mixture further comprises a splint oligonucleotide D.
In some embodiments, D comprises an oligonucleotide region complementary to the 3′ end of A1 and a region complementary to either the 5′ end of oligonucleotide C or to the 5′ end of A1.
In some embodiments, D is unable to undergo extension against A1 by virtue of either a 3′ modification or through a mismatch between the 3′ end of D and the corresponding region of A1.
In some embodiments, the method comprises the method according to any previous embodiment wherein the enzyme which performs pyrophosphorolysis of A0 to form partially digested strand A1 also amplifies A2.
In some embodiments, the method comprises the method according to any previous embodiment wherein detection is achieved using one or more oligonucleotide fluorescent binding dyes or molecular probes.
In some embodiments, an increase in signal over time resulting from the generation of amplicons of A2 is used to infer the concentration of the target sequence in the analyte.
In some embodiments, the method comprises the method according to any previous embodiment wherein multiple probes A0 are employed, each selective for a different target sequence and each including an identification region, and further characterised in that the amplicons of A2 include this identification region and therefore the target sequences present in the analyte, are inferred through the detection of the identification region(s).
In some embodiments, detection of the identification region(s) is carried out using molecular probes or through sequencing.
In some embodiments, the final step of the method further comprises the steps of:
In some embodiments, the different probes A0 comprise a common priming site, allowing a single primer or single set of primers to be used for amplification.
According to the present invention, there are provided methods of detecting the methylation status of a target polynucleotide sequence in a given nucleic acid analyte. The analytes to which the various methods of the invention can be applied may be prepared from the biological sample mentioned above by a series of preliminary steps designed to amplify the analyte and separate if from the background genomic DNA which is typically present in significant excess. This method is generally applicable to the production of single-stranded target analytes and is therefore useful in situations other than when it is integrated with or further comprises part of the method of the first aspect of the invention. Accordingly, there is provided a method for preparing at least one single-stranded analyte of a nucleic acid comprised of a target polynucleotide region characterised by the steps of (1) producing amplicons of the analyte(s) by subjecting a biological sample comprised of the analyte(s) and optionally background genomic DNA to cycles of amplification. In one preferred embodiment amplification is carried out using the polymerase chain reaction (PCR) in the presence of a polymerase, nucleoside triphosphates and at least one corresponding primer pair wherein one of the primers includes a 5′-3′ exonuclease blocking group and (2) optionally digesting the product of step (1) with an exonuclease having 5′-3′ exonucleolytic activity. In one embodiment, the method may further comprise (3) reacting the product of step (2) with a proteinase to destroy the polymerase and thereafter (4) deactivating the proteinase by heating the product of step (3) to a temperature in excess of 50° C. In one preferred embodiment steps (1) to (4) are carried out prior to step (a) of the method of the first aspect of the invention to produce an integrated method of detecting target sequences derived from a biological sample. In another embodiment, the biological sample has undergone cell lysis before step (1) is carried out.
In some embodiments of step (1) the nucleoside triphosphates are a mixture of the four deoxynucleoside triphosphates characteristic of naturally occurring DNA. In a preferred embodiment the mixture of deoxynucleoside triphosphates comprise deoxyuridine triphosphate (dUTP) instead of deoxythymidine triphosphate (dTTP) and step (1) is further carried out in the presence of the enzyme dUTP-DNA glycolase (UDG) to remove any contaminating amplicons from previous assays. In yet another embodiment, a high fidelity polymerase is used in step (1) for example one of those sold under the trade name Phusion® or Q5. In yet another embodiment, the polymerase may be KAPA HiFi Uracil+ DNA Polymerase.
High-fidelity DNA polymerases have several safeguards to protect against both making and propagating mistakes while copying DNA. Such enzymes have a significant binding preference for the correct versus the incorrect nucleoside triphosphate during polymerization. If an incorrect nucleotide does bind in the polymerase active site, incorporation is slowed due to the sub-optimal architecture of the active site complex. This lag time increases the opportunity for the incorrect nucleotide to dissociate before polymerase progression, thereby allowing the process to start again, with a correct nucleoside triphosphate. If an incorrect nucleotide is inserted, proofreading DNA polymerases have an extra line of defense. The perturbation caused by the mispaired bases is detected, and the polymerase moves the 3′ end of the growing DNA chain into a proofreading 3′→5′ exonuclease domain. There, the incorrect nucleotide is removed by the 3′→5′ exonuclease activity, whereupon the chain is moved back into the polymerase domain, where polymerization can continue.
In some embodiments, the nucleoside triphosphates are a mixture of synthetic or modified deoxynucleoside triphosphates.
In some embodiments, the nucleoside triphosphates are a mixture of the four deoxynucleoside triphosphates and synthetic or modified deoxynucleotide triphosphates.
In some embodiments, step (1) is carried out using a limited amount of primer and an excess of amplification cycles. By this means a fixed amount of amplicons is produced regardless of the initial amount of analyte. Thus the need for analyte quantification prior to subsequent steps is avoided. In another embodiment of step (1), which has the advantage of obviating the need for step (2), amplification is carried out in the presence of a primer pair where one of the two primers is present in excess of the other, resulting in generation of single-stranded amplicons once one primer is fully utilised.
In one preferred embodiment of step (2), the 5′ primer is blocked with an exonuclease blocking group selected from phosphorothioate linkages, inverted bases, DNA spacers and other oligonucleotide modifications commonly known in the art. In another embodiment the other primer in the pair has a phosphate group at its 5′end.
In some embodiments, in step (3) the proteinase employed is proteinase K and step (4) is carried out by heating to a temperature of 80 to 100° C. for up to 30 minutes. In one embodiment, in step (3) the proteinase employed is proteinase K, step (3) is carried out by heating to a temperature of 55° C. for 5 minutes and step (4) is carried out by heating to a temperature of 95° C. for 10 minutes. In another embodiment at some point after step (2) the reaction medium is treated with a phosphatase or phosphohydrolase to remove any residual nucleoside triphosphates which may be present.
In some embodiments, the target polynucleotide sequence in the analyte will be a gene or chromosomal region within the DNA or RNA of a cancerous tumour cell and will be characterised by the presence of one or more mutations; for example in the form of one or more single nucleotide polymorphisms (SNPs). Thus the invention will be useful in the monitoring of and/or treatment for disease recurrence. Patients who have been declared free of disease following treatment may be monitored over time to detect the recurrence of disease. This needs to be done non-invasively and requires sensitive detection of target sequences from blood samples. Similarly, for some cancers there are residual cancer cells that remain in a patient after treatment. Monitoring of the levels of these cells (or cell free DNA) present in the patient's blood, using the current invention, allows detection of recurrence of disease or failure of current therapy and the need to switch to an alternative.
In some embodiments, detection of the target polynucleotide sequence will allow repeated testing of patient samples during treatment of disease to allow early detection of developed resistance to therapy. For example, epidermal growth factor receptor (EGFR) inhibitors, such as gefitinib, erlotinib, are commonly used as first line treatments for non-small cell lung cancer (NSCLC). During treatment the tumour will often develop mutations in the EGFR gene (e.g T790M, C797S) which confer resistance to the treatment. Early detection of these mutations allows transfer of the patient onto alternative therapies.
In some embodiments, the target polynucleotide sequence in the analyte will be a gene or chromosomal region within the DNA or RNA of fetal origin and will be characterised by the presence of one or more mutations; for example in the form of one or more single nucleotide polymorphisms (SNPs). Thus, the invention may be used to detect mutations at very low allele fractions, at an earlier stage of pregnancy than other available testing techniques.
In another embodiment, the target polynucleotide sequence may be a gene or genomic region derived from an otherwise healthy individual but the genetic information obtained may assist in generating valuable companion diagnostic information allowing medical or therapeutic conclusions to be drawn across one or more defined groups within the human population.
In yet another embodiment, the target polynucleotide sequence may be characteristic of an infectious disease, or of resistance of an infectious disease to treatment with certain therapies; for example a polynucleotide sequence characteristic of a gene or chromosomal region of a bacterium or a virus, or a mutation therein conferring resistance to therapy.
In some embodiments, the target polynucleotide sequence may be characteristic of donor DNA. When a transplanted organ is rejected by the patient, the DNA from this organ is shed into the patient's bloodstream. Early detection of this DNA would allow early detection of rejection. This could be achieved using custom panels of donor-specific markers, or by using panels of variants known to be common in the population, some of which will be present in the donor and some in the recipient. Routine monitoring of organ recipients over time is thus enabled by the claimed method.
The success of organ transplantation can depend on the overall level of cumulative injury to the organ caused by several events in the donor. This includes age, lifestyle, ischemia/reperfusion injury (IRI) and immune response in the recipient. Research has shown that IRI causes epigenetic changes in the donor organ. The promoter region of the C3 gene becomes demethylated in the kidney, which is associated with chronic nephropathy post-transplantation. DNA methylation is a major contributor to a balanced immune response toward a graft as it regulates the function of cells of the immune system. Thus, detection of the methylation status of particular DNA sequences can allow identification of patients at risk for post-transplant complications.
In yet another embodiment, various versions of the method using different combinations of probes (see below) are employed in parallel so that the analyte can be simultaneously screened for multiple target sequences; for example sources of cancer, cancer indicators or multiple sources of infection. In this approach, the amplified products obtained in by parallel application of the method are contacted with a detection panel comprised of one or more oligonucleotide binding dyes or sequence specific molecular probes such as a molecular beacon, hairpin probe or the like. Thus, in another aspect of the invention there is provided the use of at least one probe and optionally one ligation oligonucleotide in combination with one or more chemical and biological probes selective for the target polynucleotide sequences or with the use of sequencing to identify the amplified probe regions.
In some embodiments, the single-stranded probe oligonucleotide A0 comprises a priming region and a 3′ end which is complementary to the target polynucleotide sequence to be detected. By this means, a first intermediate product is created which is at least partially double-stranded. In one embodiment, this step is carried out in the presence of excess A0 and in an aqueous medium containing the analyte and any other nucleic acid molecules.
During step (a), the double-stranded region of the first intermediate product is pyrophosphorolysed in the 3′-5′ direction from the 3′ end of its A0 strand. As a consequence, the A0 strand is progressively digested to create a partially digested strand; hereinafter referred to as A1. Where the probe oligonucleotide erroneously hybridises with a non-target sequence, the pyrophosphorolysis reaction will stop at any mismatches, preventing subsequent steps of the method from proceeding. In another embodiment, this digestion continues until A1 lacks sufficient complementarity with the analyte or a target region therein to form a stable duplex. At this point, the various strands then separate by melting, thereby producing A1 in single-stranded form. Under typical pyrophosphorolysis conditions, this separation occurs when there are between 6 and 20 complementary nucleotides between the analyte and A0.
In another embodiment, the digestion continues until A1 lacks sufficient complementarity with the analyte or target region therein for the pyrophosphorolysing enzyme to bind or for the pyrophosphorolysing reaction to continue. This typically occurs when there are between 6 and 20 complementary nucleotides remaining between the analyte and probe. In some embodiments, this occurs when there are between 6 and 40 complementary nucleotides remaining.
In another embodiment, in which a splint oligonucleotide D having complementarity to the 5′ and 3′ ends of A1 is employed (see below), the digestion continues until the length of complementarity between A1 and the target is reduced to the point at which it is energetically favourable for oligo D to displace the analyte molecule from A1. This typically occurs when the region of complementarity between A1 and the analyte molecule is of similar or shorter length than the region of complementarity between oligo D and the 3′ end of A1, but may also occur when the complementarity between A1 and the analyte molecule is longer than this due to the favourability of intra-molecular hybridisation of the oligo D, which may already be hybridised to the 5′ end of A1.
In another embodiment, in which the ligation of A1 is performed using the analyte molecule as a splint (see
Suitably, pyrophosphorolysis is carried out in the reaction medium at a temperature in the range 20 to 90° C. in the presence of at least a polymerase exhibiting pyrophosphorolysis activity and a source of pyrophosphate ion. Further information about the pyrophosphorolysis reaction as applied to the digestion of polynucleotides can be found for example in J. Biol. Chem. 244 (1969) pp. 3019-3028 or our earlier patent application.
In some embodiments, the pyrophosphorolysis step is driven by the presence of a source of excess polypyrophosphate, suitable sources including those compounds containing 3 or more phosphorous atoms.
In some embodiments, the first reaction mixture comprises a source of excess polypyrophosphate.
In some embodiments, the pyrophosphorolysis step is driven by the presence of a source of excess modified pyrophosphate. Suitable modified pyrophosphates include those with other atoms or groups substituted in place of the bridging oxygen, or pyrophosphate (or polypyrophosphate) with substitutions or modifying groups on the other oxygens. The person skilled in the art will understand that there are many such examples of modified pyrophosphate which would be suitable for use in the current invention, a non-limiting selection of which are:
In some embodiments, the first reaction mixture comprises a source of excess modified polypyrophosphate.
In one preferred embodiment, the source of pyrophosphate ion is PNP, PCP or Tripolyphoshoric Acid (PPPi).
Further, but not limiting, examples of sources of pyrophosphate ion for use in the pyrophosphorolysis step (b) may be found in WO2014/165210 and WO00/49180.
In one embodiment, the source of excess modified pyrophosphate can be represented as Y—H wherein Y corresponds to the general formula (X—O)2P(═B)—(Z—P(═B)(O—X))n— wherein n is an integer from 1 to 4; each Z— is selected independently from —O—, —NH— or —CH2—; each B is independently either O or S; the X groups are independently selected from —H, —Na, —K, alkyl, alkenyl, or a heterocyclic group with the proviso that when both Z and B correspond to —O— and when n is 1 at least one X group is not H.
In some embodiments, Y corresponds to the general formula (X—O)2P(═B)—(Z—P(═B)(O—X))n— wherein n is 1, 2, 3 or 4. In another embodiment, the Y group corresponds to the general formula (X—O)2P(═O)—Z—P(═O)(O—H)— wherein one of the X groups is —H. In yet another preferred embodiment, Y corresponds to the general formula (X—O)2P(═O)—Z—P(═O)(O—X)— wherein at least one of the X groups is selected from methyl, ethyl, allyl or dimethylallyl.
In an alternative embodiment, Y corresponds to either of the general formulae (H—O)2P(═O)—Z—P(═O)(O—H)— wherein Z is either —NH— or —CH2— or (X—O)2P(═O)—Z—P(═O)(O—X)— wherein the X groups are all either —Na or —K and Z is either —NH— or —CH2—.
In another embodiment, Y corresponds to the general formula (H—O)2P(═B)—O—P(═B)(O—H)— wherein each B group is independently either O or S, with at least one being S.
Specific examples of preferred embodiments of Y include those of the formula (X1-O)(HO)P(═O)—Z—P(═O)(O—X2) wherein Z is O, NH or CH2 and (a) X1 is γ,γ-dimethylallyl, and X2 is —H; or (b) X1 and X2 are both methyl; or (c) X1 and X2 are both ethyl; or (d) X1 is methyl and X2 is ethyl or vice versa.
In some embodiments, where molecular probes are to be used for detection, the probe oligonucleotide A0 is configured to include an oligonucleotide identification region on the 5′ side of the region complementary to the target sequence, and the molecular probes employed are designed to anneal to this identification region. In one embodiment, only the 3′ region of A0 is able to anneal to the target; i.e. any other regions lack sufficient complementarity with the analyte for a stable duplex to exist at the temperature at which the pyrophosphorolysis step is carried out. Here and throughout, by the term ‘sufficient complementarity’ is meant that, to the extent that a given region has complementarity with a given region on the analyte, the region of complementarity is more than 10 nucleotides long.
In a further aspect of the methods of the invention there is provided alternate embodiments in which the phosphorolysis step of any previous embodiment is replaced with an exonuclease digestion step using a double-strand specific exonuclease. The person skilled in the art will understand that double-strand specific exonucleases include those that read in the 3′-5′ direction, such as ExoIII, and those that read in the 5′-3′ direction, such as Lambda Exo, amongst many others.
In embodiments of the invention wherein the exonuclease digestion step utilises a double strand-specific 5′-3′ exonuclease, it is the 5′ end of A0 that is complementary to the target analyte and the common priming sequence and blocking group are located on the 3′ side of the region complementary to the target. In a further embodiment, where molecular probes are to be used for detection the probe oligonucleotide A0 is configured to include an oligonucleotide identification region on the 3′ side of the region complementary to the target sequence, and the molecular probes employed are designed to anneal to this identification region.
In embodiments of the invention wherein the exonuclease digestion step utilises a double strand-specific 5′-3′ exonuclease, an exonuclease having 3′ to 5′ exonucleolytic activity can optionally be added to the first reaction mixture for the purpose of digesting any other nucleic acid molecules present whilst leaving A0 and any material comprising partially digested strand A1 intact. Suitably, this resistance to exonucleolysis is achieved as described elsewhere in this application.
In one preferable embodiment of the invention, the 5′ end of A0 or an internal site on the 5′ side of the priming region is rendered resistant to exonucleolysis. By this means and after, or simultaneously with, the pyrophosphorolysis step, an exonuclease having 5′-3′ exonucleolytic activity can optionally be added to the reaction medium for the purpose of digesting any other nucleic acid molecules present whilst leaving A0 and any material comprising the partially digested strand A1 intact. Suitably, this resistance to exonucleolysis is achieved by introducing one or more blocking groups into the oligonucleotide A0 at the required point. In one embodiment, these blocking groups may be selected from phosphorothioate linkages and other backbone modifications commonly used in the art, C3 spacers, phosphate groups, modified bases and the like.
In some embodiments, the identification region will comprise or have embedded within a barcoding region which has a unique sequence and is adapted to be indirectly identified using a sequence-specific molecular probe applied to the amplified components A2 or directly by the sequencing of these components. Examples of molecular probes which may be used include, but are not limited to, molecular beacons, TaqMan® probes, Scorpion® probes and the like.
In some embodiments the A2 strand or a desired region thereof is caused to undergo amplification so that multiple, typically many millions, of copies are made. This is achieved by priming a region of A2 and subsequently any amplicons derived therefrom with single-stranded primer oligonucleotides, provided for example in the form of a forward/reverse or sense/antisense pair, which can anneal to a complementary region thereon. The primed strand then becomes the point of origin for amplification. Amplification methods include, but are not limited to, thermal cycling and isothermal methods such as the polymerase chain reaction, recombinase polymerase amplification and rolling circle amplification; the last of these being applicable when A2 is circularised. By any of these means, many amplicon copies of a region of A2 and in some instances its sequence complement can be rapidly created. The exact methodologies for performing any of these amplification methods will be well-known to one of ordinary skill and the exact conditions and temperature regimes employed are readily available in the general literature to which the reader is directed. Specifically, in the case of the polymerase chain reaction (PCR), the methodology generally comprises extending the primer oligonucleotide against the A2 strand in the 5′-3′ direction using a polymerase and a source of the various single nucleoside triphosphates until a complementary strand is produced; dehybridising the double-stranded product produced to regenerate the A2 strand and the complementary strand; re-priming the A2 strand and any of its amplicons and thereafter repeating these extension/dehybridisation/repriming steps multiple times to build-up a concentration of A2 amplicons to a level where they can be reliably detected.
In an alternative embodiment, the first or second reaction mixtures further comprises components for the hybridisation chain reaction (HCR).
In some embodiments, the first reaction mixture further comprises a ligation probe oligonucleotide C which has a 5′ phosphate, a splint oligonucleotide D which is complementary to the 3′ end of A1 and the 5′ end of C, and the partially digested strand A1 is ligated at the 3′ end to the 5′ end of C to form oligonucleotide A2. In some embodiments, the second reaction mixture further comprises hairpin oligonucleotide 1 (HO1) and hairpin oligonucleotide 2 (HO2), each of which comprises a fluorophore and quencher such that when each oligonucleotide remains in a hairpin configuration the fluorophore and quencher are in contact with each other. HO1 is designed such that A2 anneals to it, opening the ‘hairpin’ structure and separating the fluorophore from the quencher. The now ‘open’ HO1 is now able to anneal to HO2, opening the ‘hairpin’ structure and separating the fluorophore from the quencher.
In some embodiments, there are a plurality of hairpin oligonucleotides present such that the presence of one A2 is able to cause a chain reaction of hairpin oligonucleotide opening resulting in the generation of a detectable fluorescent signal. This methodology is known in the literature as the Hybridisation Chain Reaction (HCR).
In some embodiments, the fluorophore of the fluorophore-quencher pair is selected from, but not limited to, dyes of the fluorescein family, the carboxyrhodamine family, the cyanine family, and the rhodamine family. Other families of dyes that can be used include, e.g., polyhalofluorescein-family dyes, hexachlorofluorescein-family dyes, coumarin-family dyes, oxazine-family dyes, thiazine-family dyes, squaraine-family dyes, chelated lanthanide-family dyes, the family of dyes available under the trade designation Alexa Fluor J, from Molecular Probes, the family of dyes available under the trade designation Atto from ATTO-TEC (Siegen, Germany) and the family of dyes available under the trade designation Bodipy J, from Invitrogen (Carlsbad, Calif.). Dyes of the fluorescein family include, e.g., 6-carboxyfluorescein (FAM), 2′,4′,1,4,-tetrachlorofluorescein (TET), 2′,4′,5′,7′,1,4-hexachlorofluorescein (HEX), 2′,7′-dimethoxy-4′,5′-dichloro-6-carboxyrhodamine (JOE), 2′-chloro-5′-fluoro-7′,8′-fused phenyl-1,4-dichloro-6-carboxyfluorescein (NED), 2′-chloro-7′-phenyl-1,4-dichloro-6-carboxyfluorescein (VIC), 6-carboxy-X-rhodamine (ROX), and 2′,4′,5′,7′-tetrachloro-5-carboxy-fluorescein (ZOE). Dyes of the carboxyrhodamine family include tetramethyl-6-carboxyrhodamine (TAMRA), tetrapropano-6-carboxyrhodamine (ROX), Texas Red, R110, and R6G. Dyes of the cyanine family include Cy2, Cy3, Cy3.5, Cy5, Cy5.5, and Cy7. Fluorophores are readily available commercially from, for instance, Perkin-Elmer (Foster City, Calif.), Molecular Probes, Inc. (Eugene, Oreg.), and Amersham GE Healthcare (Piscataway, N.J.).
In some embodiments, the quencher of the fluorophore-quencher pair may be a fluorescent quencher or a non-fluorescent quencher. Fluorescent quenchers include, but are not limited to, TAMRA, ROX, DABCYL, DABSYL, cyanine dyes including nitrothiazole blue (NTB), anthraquinone, malachite green, nitrothiazole, and nitroimidazole compounds. Exemplary non-fluorescent quenchers that dissipate energy absorbed from a fluorophore include those available under the trade designation Black Hole™ from Biosearch Technologies, Inc. (Novato, Calif.), those available under the trade designation Eclipse™. Dark, from Epoch Biosciences (Bothell, Wash.), those available under the trade designation Qx1J, from Anaspec, Inc. (San Jose, Calif.), those available under the trade designations ZEN and TAO from Integrated DNA Technologies (Coralville, Iowa) and those available under the trade designation Iowa Black™ from Integrated DNA Technologies (Coralville, Iowa).
In some embodiments, the fluorophore of the fluorophore-quencher pair may be fluorescein, Lucifer Yellow, B-phycoerythrin, 9-acridineisothiocyanate, Lucifer Yellow VS, 4-acetamido-4′-isothio-cyanatostilbene-2,2′-disulfonic acid, 7-diethylamino-3-(4′-isothiocyanatophenyl)-4-methylcoumarin, succinimdyl 1-pyrenebutyrate, and 4-acetamido-4′-isothiocyanatostilbene-2-,2′-disulfonic acid derivatives.
In some embodiments, the fluorophore of the fluorophore-quencher pair may be LC-Red 640, LC-Red 705, Cy5, Cy5.5, Lissamine rhodamine B sulfonyl chloride, tetramethyl rhodamine isothiocyanate, rhodamine x isothiocyanate, erythrosine isothiocyanate, fluorescein, diethylenetriamine pentaacetate or other chelates of Lanthanide ions (e.g., Europium, or Terbium).
In some embodiments, the invention utilises fluorescently labelled oligonucleotides that are double quenched. The inclusion of a second, internal quencher shortens the distance between the dye and quencher and, in concert with the first quencher, provides greater overall dye quenching, lowering background and increasing signal detection. The second and first quenchers may be any of the quenchers previously described.
In some embodiments, the first reaction mixture further comprises a ligation probe oligonucleotide C which has a 5′ phosphate, a splint oligonucleotide D which is complementary to the 3′ end of A1 and the 5′ end of C, and the partially digested strand A1 is ligated at the 3′ end to the 5′end of C to form oligonucleotide A2.
In some embodiments, the 5′ and 3′ ends of A1 are ligated together to form a circularised A2.
In some embodiments, A1 is circularised to form A2 against a ligation probe oligonucleotide C.
In some embodiments, A1 is circularised to form A2 against the target sequence.
In some embodiments, A1 is circularised to form A2 as previously or subsequently, described.
In some embodiments, A2 is formed from partially digested strand A1 as previously, or subsequently, described.
In some embodiments, the second reaction mixture further comprises:
In some embodiments, the fluorophore-quencher pair may be as described previously. In some embodiments, the first and second reaction mixtures are combined.
In some embodiments, the first and second reactions mixtures are combined such that pyrophosphorolysis, ligation and the generation of a detectable fluorescent signal occurs without the addition of further reagents.
In an alternative embodiment, the second reaction mixture further comprises one or more DNAzymes.
In some embodiments, the second reaction mixture further comprises a partially double-stranded nucleic acid construct wherein:
In some embodiments, the fluorophore-quencher pair may be as described previously.
In some embodiments, further reagents such as suitable buffers and/or ions are present in the second reaction mixture.
In some embodiments, the second reaction mixture further comprises Mg2+ ions.
In some embodiments, the second reaction mixture further comprises Zn2+ ions.
In some embodiments, the second reaction mixture further comprises X2+ ions, wherein X is a metal.
In some embodiments, the second reaction mixture further comprises one or more X2+ ions, wherein X is a metal.
In an alternative embodiment, the first reaction mixture further comprises reagents for the ligase chain reaction (LCR).
In such an embodiment, the first reaction mixture further comprises:
In some embodiments, in the presence of A2 the two LCR probes will successfully anneal to A2 and be ligated together to form one oligonucleotide molecule which subsequently acts as a new target for second-round covalent ligation, leading to geometric amplification of the target of interest, in this case A2. The ligated products, or amplicons, are complementary to A2 and function as targets in the next cycle of amplification. Thus, exponential amplification of the specific target DNA sequences is achieved through repeated cycles of denaturation, hybridization, and ligation in the presence of excess LCR probes. From this, the presence of A2 and hence the target polynucleotide sequence is inferred.
In some embodiments, in the presence of A2 the two PCR probes will successfully anneal to A2 and be ligated together to form one oligonucleotide molecule which then acts as a new target for second-round covalent ligation, leading to geometric amplification of the target of interest, in this case A2, which is then detected.
In some embodiments, the ligated oligonucleotide molecule is detected in real time using an intercalating dye or molecular probe.
In some embodiments, the ligated oligonucleotide molecule is detected using gel electrophoresis.
The person skilled in the art will appreciate there are numerous techniques which would allow the detection of the ligated oligonucleotide molecule.
In some embodiments, the dNTPs are hot start dNTPs.
In some embodiments, the one or more ligases are thermostable.
In some embodiments, the one or more ligases are naturally occurring.
In another embodiment, the one or more ligases are engineered.
In some embodiments, the one or more ligases are selected from any ligase disclosed previously or subsequently.
In some embodiments, the one or more polymerases are thermostable.
In some embodiments, the one or more polymerases are selected from any polymerase disclosed previously or subsequently.
In some embodiments, the one or more polymerases are naturally occurring.
In some embodiments, the one or more polymerases are engineered.
In some embodiments, the one or more polymerases are the same as that used for the pyrophosphorolysis.
In some embodiments, one or more enzymes of the current invention are hot start enzymes.
In some embodiments, one or more enzymes of the current invention are thermostable.
In an alternative embodiment, it is a second reaction mixture, to which the products of step (a) are introduced prior to step (b), which further comprises reagents for the ligase chain reaction (LCR).
In this embodiment, the second reaction mixture comprises
In some embodiments, in the presence of A2 the two LCR probes will successfully anneal to A2 and be ligated together to form one oligonucleotide molecule which subsequently acts as a new target for second-round covalent ligation, leading to geometric amplification of the target of interest, in this case A2. The ligated products, or amplicons, are complementary to A2 and function as targets in the next cycle of amplification. Thus, exponential amplification of the specific target DNA sequences is achieved through repeated cycles of denaturation, hybridization, and ligation in the presence of excess LCR probes. From this, the presence of A2 and hence the target polynucleotide sequence is inferred.
In some embodiments, in the presence of A2 the two PCR probes will successfully anneal to A2 and be ligated together to form one oligonucleotide molecule which then acts as a new target for second-round covalent ligation, leading to geometric amplification of the target of interest, in this case A2, which is then detected.
In some embodiments, the ligated oligonucleotide molecule is detected in real time using an intercalating dye or molecular probe.
In some embodiments, the ligated oligonucleotide molecule is detected using gel electrophoresis.
The person skilled in the art will appreciate there are numerous techniques which would allow the detection of the ligated oligonucleotide molecule.
In some embodiments, the dNTPs are hot start dNTPs.
In some embodiments, the one or more ligases are thermostable.
In some embodiments, the one or more ligases are naturally occurring.
In another embodiment, the one or more ligases are engineered.
In some embodiments, the one or more ligases are selected from any ligase disclosed previously or subsequently.
In some embodiments, the one or more polymerases are thermostable.
In some embodiments, the one or more polymerases are selected from any polymerase disclosed previously or subsequently.
In some embodiments, the one or more polymerases are naturally occurring.
In another embodiment, the one or more polymerases are engineered.
In some embodiments, the one or more polymerases are the same as that used for the pyrophosphorolysis.
In some embodiments, the second reaction mixture further comprises a pyrophosphorolysing enzyme in addition to one or more polymerases.
In some embodiments, one or more enzymes of the current invention are hot start enzymes.
In some embodiments, one or more enzymes of the current invention are thermostable.
In an alternative embodiment, the first reaction mixture further comprises:
In some embodiments, the fluorophores and quenchers may be as described previously.
In some embodiments, the double strand specific DNA digestion enzyme is an exonuclease. In another embodiment, it is a polymerase with proofreading activity. In another embodiment, the second reaction mixture comprises a mixture of one or more of: an exonuclease or a polymerase with proofreading activity.
In some embodiments, the double strand specific DNA digestion enzyme is a hot start enzyme.
In some embodiments, the double strand specific DNA digestion enzyme has reduced activity at the temperature at which the pyrophosphorolysis reaction of the method takes place.
In some embodiments, the double strand specific DNA digestion enzyme has no activity at the temperature at which the pyrophosphorolysis reaction of the method takes place.
In an alternate embodiment, the second reaction mixture further comprises:
In some embodiments, the fluorophores and quenchers may be as described previously.
In some embodiments, the double strand specific DNA digestion enzyme is an exonuclease. In another embodiment, it is a polymerase with proofreading activity. In another embodiment, the second reaction mixture comprises a mixture of one or more of: an exonuclease or a polymerase with proofreading activity.
In some embodiments, the double strand specific DNA digestion enzyme is a hot start enzyme.
In some embodiments, the double strand specific DNA digestion enzyme has reduced activity at the temperature at which the pyrophosphorolysis reaction of the method takes place.
In some embodiments, the double strand specific DNA digestion enzyme has no activity at the temperature at which the pyrophosphorolysis reaction of the method takes place.
In an alternate embodiment, the second reaction mixture further comprises:
In some embodiments, the fluorophores and quenchers may be as described previously.
In some embodiments, the double strand specific DNA digestion enzyme is an exonuclease. In another embodiment, it is a polymerase with proofreading activity. In another embodiment, the second reaction mixture comprises a mixture of one or more of: an exonuclease or a polymerase with proofreading activity.
In some embodiments, the double strand specific DNA digestion enzyme is a hot start enzyme.
In some embodiments, the double strand specific DNA digestion enzyme has reduced activity at the temperature at which the pyrophosphorolysis reaction of the method takes place.
In some embodiments, the double strand specific DNA digestion enzyme has no activity at the temperature at which the pyrophosphorolysis reaction of the method takes place.
In some embodiments, the first or second reaction mixtures further comprise one or more partially double stranded DNA constructs wherein each construct contains one or more fluorophores and one or more quenchers. In some embodiments, when construct is partially double-stranded the one or more fluorophores and one or more quenchers are located in close enough proximity to each other such that sufficient quenching of the one or more fluorophores occurs.
In some embodiments, the construct is one strand of DNA with a self-complementary region that is looped back on itself.
In some embodiments, the construct comprises one primer of a primer pair.
In some embodiments, the first or second reaction mixture further comprises the other primer of a primer pair.
In some embodiments, a portion of the single stranded section of the construct hybridises to A2 and is extended against it by a DNA polymerase. In some embodiments, the other primer of the primer pair then hybridises to the extended construct. This primer is then extended against the construct, displacing the self-complementary region. Thus, the one or more fluorophores and one or more dyes are separated sufficiently for a fluorescent signal to be detected, indicating the presence of A2 in the reaction mixture.
In such an embodiment the construct may be known as a Sunrise Primer.
In some embodiments, the construct comprises two separate DNA strands.
In some embodiments, a portion of the single stranded section of the construct hybridises to A2 and is extended against it by a DNA polymerase. In some embodiments, the other primer of the primer pair then hybridises to the extended construct This primer is then extended against the construct, in the direction of the double stranded section, displacing the shorter of the DNA strands and thus the one or more fluorophores and one or more dyes are separated sufficiently for a fluorescent signal to be detected, indicating the presence of A2 in the reaction mixture.
In such an embodiment the construct may be known as a Molecular Zipper.
The person skilled in the art will appreciate that for both the Sunrise Primer and Molecular Zipper it is possible for the one or more fluorophores and the one or more quencher pairs to be located at various positions within each respective construct. The key feature is that each pair is located in sufficient proximity to one another that in the absence of A2, i.e. when no extension and strand displacement has occurred, no fluorescent signal is emitted.
In some embodiments, prior to step (a) of the method, any RNA present in the sample is transcribed into DNA.
In some embodiments, this is achieved via the use of a reverse transcriptase and appropriate nucleotides.
In some embodiments, the transcription of any RNA present in the sample into DNA occurs at the same time as any pre-amplification via PCR of nucleic acids present in the sample.
In some embodiments, the transcription of any RNA present in the sample into DNA occurs in a separate step to any pre-amplification via PCR of nucleic acids present in the sample.
In some embodiments of any of the previously, or subsequently, described methods, RNA present in the sample is not transcribed to DNA. In such an embodiment, A0 undergoes pyrophosphorolysis against an RNA sequence to form partially digested strand A1 and the method then proceeds as previously, or subsequently, described.
In some embodiments of any of the previously, or subsequently, described methods, one or more reaction mixtures may be combined.
In some embodiments, the first reaction mixture further comprises a ligation probe oligonucleotide C, and the partially digested strand A1 is ligated at the 3′ end to the 5′ end of C, while in another embodiment, A1 is circularised through ligation of its 3′ and 5′ ends;
in each case to create an oligonucleotide A2.
In one embodiment, the ligation of A1 occurs:
In some embodiments, A1 is optionally extended in 5′-3′ direction prior to ligation.
In some embodiments, this optional extension and the ligation are performed against the target oligonucleotide, while in another embodiment they are performed through addition of a further splint oligonucleotide D to which A1 anneals prior to extension and/or ligation. In one embodiment, D comprises an oligonucleotide region complementary to the 3′ end of A1 and a region complementary to either the 5′ end of oligonucleotide C or to the 5′ end of A1. In another embodiment, D is unable to extend against A1 by virtue of either a 3′-end modification or through a nucleotide mismatch between the 3′ end of D and the corresponding region of A1.
In some embodiments, the ligation probe C has a 5′ region complementary to at least part of a 5′ end region of a splint oligonucleotide D or to the target oligonucleotide. By such means, a second intermediate product is formed in which the A2 strand is comprised of A1, C and optionally an intermediate region formed by extension of A1 in the 5′-3′ direction to meet the 5′ end of C. In such an embodiment, the primers employed in step (c) (see below) are chosen to amplify at least a region of A2 including the site at which ligation of the A1 to C has occurred. In this embodiment, we have found that it is advantageous to include a 3′ blocking group on C so that a 3′-5′ exonuclease can be used to digest any non-ligated A1 prior to amplification. Suitable polymerases which may be used for the extension of A1 prior to ligation include but are not limited to Hemo KlenTaq, Mako and Stoffel Fragment.
In some embodiments, the first reaction mixture further comprises a phosphatase or phosphohydrolase to remove by hydrolysis the nucleoside triphosphates produced by the pyrophosphorolysis reaction thereby ensuring that the pyrophosphorolysis reaction can continue and does not become out-competed by the forward polymerisation reaction.
In some embodiments, prior to or during step (b) the products of the previous step are treated with a pyrophosphatase to hydrolyse the pyrophosphate ion, preventing further pyrophosphorolysis from occurring and favouring the forward polymerisation reaction.
In some embodiments, prior to or during step (b) the products of the previous step are treated with an exonuclease.
In some embodiments, the enzyme which performs pyrophosphorolysis of A0 to form partially digested strand A1 also amplifies A2. The person skilled in the art will appreciate there exists many such enzymes.
The amplicons are detected and the information obtained are used to infer whether the polynucleotide target sequence is present or absent in the original analyte and/or a property associated therewith. For example, by this means a target sequence characteristic of a cancerous tumour cell may be detected with reference to specific SNPs being looked for. As a further example, a target sequence characteristic of a cancerous tumour cell may be detected with reference to specific methylation sites being looked for. In another embodiment, a target sequence characteristic of the genome of a virus of bacterium (including new mutations thereof) may be detected. Many methods of detecting the amplicons or identification regions can be employed including for example an oligonucleotide binding dye, a sequence-specific molecular probe such as fluorescently-labelled molecular beacon or hairpin probe. Alternatively, direct sequencing of the A2 amplicons can be performed using one of the direct sequencing methods employed or reported in the art. Where oligonucleotide binding dyes, fluorescently labelled beacons or probes are employed it is convenient to detect the amplicons using an arrangement comprising a source of stimulating electromagnetic radiation (laser, LED, lamp etc.) and a photodetector arranged to detect emitted fluorescent light and to generate therefrom a signal comprising a data stream which can be analysed by a microprocessor or a computer using specifically-designed algorithms.
In some embodiments, detection is achieved using one or more oligonucleotide fluorescent binding dyes or molecular probes. In such embodiments, an increase in signal over time resulting from the generation of amplicons of A2 is used to infer the concentration of the target sequence in the analyte. In one embodiment that the final step of the method further comprises the steps of:
In some embodiments, multiple probes A0 are employed, each selective for a different target sequence and each including an identification region, and further characterised in that the amplicons of A2 include this identification region and therefore the target sequences present in the analyte are inferred through the detection of the identification region(s). In such embodiments detection of the identification regions(s) is carried out using molecular probes or through sequencing.
In some embodiments, is the one or more nucleic acid analytes are split into multiple reaction volumes, each volume having a one or more probe oligonucleotide A0, introduced to detect different target sequences.
In some embodiments wherein different probes A0 are used the different probes A0 comprise one or more common priming sites, allowing a single primer or single set of primers to be used for amplification.
In another aspect of the invention, there is provided a method of identifying a target polynucleotide sequence in a given nucleic acid analyte characterised by the steps of any previous embodiment of the invention wherein the multiple copies of A2, or a region of A2, are labelled using one or more oligonucleotide fluorescent binding dyes or molecular probes. The fluorescent signal of these multiple copies is measured and the multiple copies are exposed to a set of denaturing conditions. The target polynucleotide sequence is the identified by monitoring a change in the fluorescent signal of the multiple copies during exposure to the denaturing conditions.
In some embodiments, the denaturing conditions may be provided by varying the temperature e.g. increasing the temperature to a point where the double strand begins to dissociate.
Additionally or alternatively, the denaturing conditions may also be provided by varying the pH such that the conditions are acidic or alkaline, or by adding in additives or agents such as a strong acid or base, a concentrated inorganic salt or organic solvent e.g. alcohol.
In another aspect of the invention, there is provided the use of the methods described above to screen mammalian subjects, especially human patients, for the presence of infectious diseases, cancer or for the purpose of generating companion diagnostic information.
In a further aspect of the invention, there is provided control probes for use in the methods as described above. Embodiments of the current invention include those wherein the presence of a specific target sequence, or sequences, is elucidated by the generation of a fluorescent signal. In such embodiments, there may inevitably be a level of signal generated from non-target DNA present in the sample. For a given sample, this background signal has a later onset than the ‘true’ signal, but this onset may vary between samples. Accurate detection of the presence of low concentrations of target sequence, or sequences, thus relies on knowledge of what signal is expected in its absence. For contrived samples references are available, but for true ‘blind’ samples from patients this is not the case. The control probes (E0) are utilised to determine the expected background signal profile for each assay probe. The control probe targets a sequence not expected to be present in the sample and the signal generated from this probe can then be used to infer the expected rate of signal generation from the sample in the absence of target sequence.
Thus there is provided a method of detecting a target polynucleotide sequence in a given nucleic acid analyte according to any of the previously described methods characterised by the steps of:
In some embodiments, the control probe (E0) and A0 are added to separate portions of the sample while in another embodiment the E0 and A0 are added to the same portion of the sample and different detection channels (e.g. different colour dyes) used to measure their respective signals. The signal generated by E0 may then be utilised to infer and correct for the background signal expected to be generated by A0 in the absence of the polynucleotide target sequence in the sample. For example, a correction of the background signal may involve the subtraction of the signal observed from E0 from that observed from A0, or through the calibration of the signal observed from A0 using a calibration curve of the relative signals generated by A0 and E0 under varying conditions.
In some embodiments, a single E0 can be used to calibrate all of the assay probes which may be produced.
In some embodiments, a separate E0 may be used to calibrate each amplicon of the sample DNA generated in an initial amplification step. Each amplicon may contain multiple mutations/target sequences of interest, but a single E0 will be sufficient to calibrate all of the assay probes against a single amplicon.
In a further embodiment, a separate E0 may be used for each target sequence. For example, if a C>T mutation is being targeted, an E0 could be designed that targets a C>G mutation in the same site that is not known to occur in patients. The signal profile generated by E0 under various conditions can be assessed in calibration reactions and these data used to infer the signal expected from the assay probe targeting the C>T variant when this variant is not present.
The specificity of the methods of the current invention may be improved by the introduction of blocking oligonucleotides. For example a blocking oligo nucleotide can be introduced so as to hybridise to at least a portion of wild-type DNA, promoting annealing of A0 only to the target polynucleotide sequences and not the wildtype. Alternatively or additionally, blocking oligonucleotides can be used to improve the specificity of the polymerase chain reaction (PCR) to prevent amplification of any wild type sequence present. The general technique used is to design an oligonucleotide that anneals between the PCR primers and is not able to be displaced or digested by the PCR polymerase. The oligonucleotide is designed to anneal to the non-target (usually healthy) sequence, while being mismatched (often by a single base) to the target (mutant) sequence. This mismatch results in a different melting temperature against the two sequences, the oligonucleotide being designed to remain annealed to the non-target sequence at the PCR extension temperature while dissociating from the target sequence.
The blocking oligonucleotides may often have modifications to prevent its digestion by the exonuclease activity of the PCR polymerase, or to enhance the melting temperature differential between the target and non-target sequence.
The incorporation of a locked nucleic acid (LNA) or other melting temperature altering modification into a blocking oligonucleotide can significantly increase the differential in melting temperature of the oligonucleotide against target and non-target sequences.
Thus there is provided an embodiment of the invention wherein blocking oligonucleotides are used. The blocking oligonucleotides must be resistant to the pyrophosphorolysing (PPL) reaction to ensure they are not digested or displaced. This can be achieved in a number of different ways, for example via mismatches at their 3′ ends or through modifications such as phosphorothioate bonds or spacers.
In such embodiments or an aspect of the present invention where blocking oligonucleotides are used, the method of detecting a target polynucleotide sequence in a given nucleic acid analyte is characterised by annealing single-stranded blocking oligonucleotides to at least a subset of non-target polynucleotide sequences before, or during, the same step wherein the analyte target sequence is annealed to a single-stranded probe oligonucleotide A0 to create a first intermediate product which is at least partially double-stranded and in which the 3′ end of A0 forms a double-stranded complex with the analyte target sequence.
In some embodiments, the blocking oligonucleotides are made to be resistant to the pyrophosphorolysing reaction via mismatches at their 3′ ends. In another embodiment, the blocking oligonucleotides are made to be resistant via the presence of a 3′-blocking group. In another embodiment the blocking oligonucleotides are made to be resistant via the presence of spacers or other internal modifications. In a further embodiment the blocking oligonucleotides include both a melting temperature increasing modification or modified nucleotide base and are rendered resistant to pyrophosphorolysis.
References herein to ‘phosphatase enzymes’ refer to any enzymes, or functional fragments thereof, with the ability to remove by hydrolysis the nucleoside triphosphates produced by the methods of the current invention. This includes any enzymes, or functional fragments thereof, with the ability to cleave a phosphoric acid monoester into a phosphate ion and an alcohol.
References herein to ‘pyrophosphatase enzymes’ refer to any enzymes, or functional fragments thereof, with the ability to catalyse the conversion of one ion of pyrophosphate to two phosphate ions.
This also includes inorganic pyrophosphatases and inorganic diphosphatases. A non-limiting example is thermostable inorganic pyrophosphate (TIPP).
In some embodiments there is provided a modified version of any previously described embodiment wherein the use of a pyrophosphatase is optional.
In some embodiments of the invention there is provided a kit comprising:
In some embodiments of the invention there is provided a kit for use in a method of detecting a target polynucleotide sequence in a given nucleic acid analyte present in a sample, comprising:
In some embodiments, the kit comprises
In some embodiments, the kit may alternatively further comprise:
In some embodiments, the kit may alternatively further comprise:
In some embodiments, the kit may further comprise:
In some embodiments, the kit may further comprise a plurality of HO1 and HO2.
In some embodiments, the kit may alternatively further comprise an oligonucleotide A comprising a substrate arm, a partial catalytic core and a sensor arm;
In some embodiments, the kit may alternatively further comprise a partially double-stranded nucleic acid construct wherein:
In some embodiments, the kit may further comprise an enzyme for removal of the at least one RNA base.
In some embodiments, the enzyme is Uracil-DNA Glycosylase (UDG) and the RNA base is uracil.
In some embodiments, the kit may alternatively further comprise:
In some embodiments, the double strand specific DNA digestion enzyme is an exonuclease.
In some embodiments, the double strand specific DNA digestion enzyme is a polymerase with proofreading activity.
In some embodiments, the fluorophore of the kit may be selected from dyes of the fluorescein family, the carboxyrhodamine family, the cyanine family, the rhodamine family, polyhalofluorescein-family dyes, hexachlorofluorescein-family dyes, coumarin-family dyes, oxazine-family dyes, thiazine-family dyes, squaraine-family dyes, and chelated lanthanide-family dyes.
In some embodiments, the fluorophore of the kit may be selected from any of the commercially available dyes or any previously or subsequently described dyes.
In some embodiments, the quencher of the kit may be selected from those available those available under the trade designations Black Hole™, Eclipse™. Dark, Qx1J, and Iowa Black™.
In some embodiments, the quencher of the kit may be selected from any of the commercially available quenchers or any previously or subsequently described quenchers.
In some embodiments, the kit further comprises a phosphatase or a phosphohydrolase.
In some embodiments, the kit further comprises a pyrophosphatase.
In some embodiments, the kit further comprises an enzyme for the transcription of RNA into DNA.
In some embodiments of the kit, the enzyme is a reverse transcriptase.
In some embodiments of the kit, one or more enzymes are hot start.
In some embodiments of the kit, one or more enzymes are thermostable.
In some embodiments, the kit may further comprise suitable washing and buffer reagents.
In some embodiments the kit further comprises a source of pyrophosphate ion.
Suitable source(s) of pyrophosphate ion are as described previously.
In some embodiments, the kit further comprises suitable positive and negative controls.
In some embodiments, the kit may further comprise one or more control probes (E0) which are as previously described.
In some embodiments, the kit may further comprise one or more blocking oligonucleotides which are as previously described.
In some embodiments, the kit may further comprises one or more control probes (E0) and one or more blocking oligonucleotides.
In some embodiments, the 5′ end of A0 may be rendered resistant to 5′-3′ exonuclease digestion and the kit may further comprise a 5′-3′ exonuclease.
In some embodiments a kit may further comprise a ligation probe oligonucleotide C.
In some embodiments a kit may further comprise a splint oligonucleotide D.
In some embodiments, a kit may comprise both C and D.
The ligation probe C may comprise a 3′ or internal modification protecting it from 3′-5′ exonuclease digestion.
D may comprise an oligonucleotide region complementary to the 3′ end of A1 and a region complementary to either the 5′ end of oligonucleotide C or the 5′ end of A1.
In some embodiments, D may be unable to undergo extension against A1 by virtue of either a 3′ modification or through a mismatch between the 3′ end of D and the corresponding region of A1 or C.
In some embodiments, the kit may further comprise dNTPs, a polymerase and suitable buffers for the initial amplification of a target polynucleotide sequence present in a sample.
In some embodiments, the kit may further comprise a dUTP incorporating high fidelity polymerase, dUTPs and uracil-DNA N-glycosylase (UDG).
In some embodiments, the kit may further comprise a phosphatase or a phosphohydrolase.
In some embodiments, the kit may further comprise a pyrophosphatase. The pyrophosphatase may be hot start.
In some embodiments, the kit may further comprise a proteinase.
In some embodiments, the kit may further comprise one or more oligonucleotide binding dyes or molecular probes.
In some embodiments, the kit may further comprises multiple A0, each selective for a different target sequence and each including an identification region.
In some embodiments, the amplification enzyme, of (e), and the pyrophosphorolysing enzyme are the same.
In some embodiments, the kit may further comprise a restriction endonuclease.
In some embodiments, the kit may further comprise a restriction endonuclease that recognises a sequence that is created by the conversion (chemically or enzymatically) of unmethylated cytosine bases in a polynucleotide sequence.
In some embodiments, the kit may further comprises a restriction endonuclease that recognises a sequence that is removed by the conversion (chemically or enzymatically) of unmethylated cytosine bases in a polynucleotide sequence.
In some embodiments, the kit may further comprise a methylation-sensitive or methylation-dependent restriction endonuclease.
In some embodiments, the restriction endonuclease recognises only sequences in which a target methylation state is not present.
In some embodiments, the kit may further comprise an epigenetic-sensitive or epigenetic-dependent restriction endonuclease, which may be as previously described.
In some embodiments, the kit may further comprises reagents for methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) of methylated DNA.
In some embodiments, the kit may further comprise components suitable for methylated DNA immunoprecipitation (MeDIP).
In some embodiments, the kit may further comprise methyl-binding proteins. In one embodiment, the methyl-binding proteins are selected from one or more of MBD2b or the MBD2b/MBD3L1 complex.
The kit may further comprise purification devices and reagents for isolating and/or purifying a portion of polynucleotides, following treatment as described herein. Suitable reagents are well known in the art and include gel filtration columns and washing buffers. Further examples of suitable reagents include magnetic beads and column filtration reagents. In one embodiment, more than one reagent for isolating and/or purifying a portion of polynucleotides are present in the kit.
In some embodiments of the invention there is provided a device comprising:
In some embodiments, the pyrophosphorolysis enzyme which was present in the wells of the second region is carried through to the wells of third region wherein it performs amplification of A2 in the presence of dNTPs and suitable buffers.
In some embodiments, a means for detecting a signal is located within one or more wells of the third region.
In some embodiments, a means for detecting a signal is located within the third region of the device.
In some embodiments, a means for detecting a signal is located within an adjacent region of the device.
In some embodiments, the dNTPs of each well of the first region may be dUTP, dGTP, dATP and dCTP and each well may further comprise a dUTP incorporating high fidelity polymerase and uracil-DNA N-glycosylase (UDG).
In some embodiments, each well of the second region may further comprise a source of pyrophosphate ion.
In some embodiments, the 5′ end of A0 may be rendered resistant to 5′-3′ exonuclease digestion and the wells of the second region may further comprises a 5′-3′ exonuclease.
In some embodiments, each well of the second or third regions may further comprise a ligase.
In some embodiments, each well of the second or third regions may further comprise a ligase and a ligation probe oligonucleotide C or a splint oligonucleotide D.
The ligation probe C may comprise a 3′ or internal modification protecting it from 3′-5′ exonuclease digestion.
The splint oligonucleotide D may comprise an oligonucleotide region complementary to the 3′ end of A1 and a region complementary to either the 5′ end of oligonucleotide C or to the 5′ end of A1.
D may be unable to undergo extension against A1 by virtue of either a 3′ modification or through a mismatch between the 3′ end of D and the corresponding region of A1 or C.
In some embodiments, the dNTPs may be hot start.
In some embodiments, each well of the second region may further comprise a phosphatase or a phosphohydrolase.
In some embodiments, each well of the second region may further comprise a pyrophosphatase.
In some embodiments, the pyrophosphatase is hot start.
In some embodiments, each well of the third region may further comprise one or more oligonucleotide binding dyes or molecular probes.
In some embodiments, each well of the second region may comprise at least one or more different A0 that is selective for a target sequence including an identification region.
In some embodiments, the amplification enzyme and the pyrophosphorolysing enzyme in the second region may be the same.
In some embodiments, there may be a fourth region comprising one or more wells, wherein each well may comprise a proteinase and wherein said fourth region may be located between the first and second regions.
In some embodiments, the second and third regions of the device may be combined such that the wells of the second region further comprise:
In some embodiments, the second and third regions of the device may be combined such that the wells of the second region further comprise:
In some embodiments, the pyrophosphorolysis enzyme which was present in the wells of the second region is utilised to perform amplification of A2 in the presence of dNTPs and suitable buffers.
In some embodiments, a means for detecting a signal is located within one or more wells of the second region.
In some embodiments, a means for detecting a signal is located within the second region of the device.
In some embodiments, a means for detecting a signal is located within an adjacent region of the device.
In some embodiments, the first region may be fluidically connected to a sample container via a fluidic interface.
In some embodiments of the invention there is provided a device comprising:
In some embodiments, the means for selectively modifying a nucleic acid may be chemicals capable of converting unmodified cytosine bases in a target polynucleotide sequence.
In some embodiments, the means for selectively modifying a nucleic acid may be enzymes capable of converting unmodified cytosine bases in a target polynucleotide sequence.
In some embodiments, the wells of the second or third region may further comprise a restriction endonuclease.
In some embodiments, located between the first and second region may be a region comprising one or more wells wherein each well may comprise a restriction endonuclease.
In some embodiments, the restriction endonuclease may recognise a sequence in a target polynucleotide sequence created by chemical or enzymatic conversion of unmodified cytosine bases.
In some embodiments, the sequence in a target polynucleotide sequence which the restriction endonuclease may recognise is removed by chemical or enzymatic conversion of unmodified cytosine bases.
In some embodiments, the restriction endonuclease may be a methylation-sensitive or methylation-dependent restriction endonuclease.
In some embodiments, the wells of the second region may comprise reagents for modification-specific multiplex ligation-dependent probe amplification (MS-MLPA) of epigenetically modified DNA.
In some embodiments, located between the first and second region may be a region comprising one or more wells wherein each well may comprise reagents for PCR.
In some embodiments, located between the first and second region may be a region comprising one or more wells wherein each well may comprise reagents for reduction of a population of epigenetically modified or unmodified target sequences.
In some embodiments, the reagents for reduction of a population of epigenetically modified or unmodified target sequences are reagents for epigenetically modified DNA immunoprecipitation, optionally methylated DNA immunoprecipitation (MeDIP).
In some embodiments, the reagents for reduction of a population of epigenetically modified or unmodified target sequences are methyl-binding proteins, such as MBD2b or the MBD2b/MBD3L1 complex.
In some embodiments, the reagents for reduction of a population of epigenetically modified or unmodified target sequences are located within one or more wells of the first region.
In some embodiments of the device, the epigenetic modification may be methylation. In some embodiments, it may be methylation at CpG islands. In some embodiments, it may be hydroxymethylation at CpG islands.
In some embodiments, the wells of the second, third or fourth region may comprise:
In some embodiments, the dNTPs of each well may be dUTP, dGTP, dATP and dCTP and each well may further comprise a dUTP incorporating high fidelity polymerase and uracil-DNA N-glycosylase (UDG).
In some embodiments, each well may further comprise a source of pyrophosphate ion.
In some embodiments, the 5′ end of A0 may be rendered resistant to 5′-3′ exonuclease digestion and the wells of the second or third region may further comprise a 5′-3′ exonuclease.
In some embodiments, each well of the third or fourth regions may further comprise a ligase.
In some embodiments, each well of the third or fourth regions may further comprise a ligase and a ligation probe oligonucleotide C or a splint oligonucleotide D.
The ligation probe C may comprise a 3′ or internal modification protecting it from 3′-5′ exonuclease digestion.
The splint oligonucleotide D may comprise an oligonucleotide region complementary to the 3′ end of A1 and a region complementary to either the 5′ end of oligonucleotide C or to the 5′ end of A1.
D may be unable to undergo extension against A1 by virtue of either a 3′ modification or through a mismatch between the 3′ end of D and the corresponding region of A1 or C.
In some embodiments, the dNTPs may be hot start.
In some embodiments, each well of the third region may further comprise a phosphatase or a phosphohydrolase.
In some embodiments, each well of the third region may further comprise a pyrophosphatase.
In some embodiments, each well of the fourth region may further comprise a pyrophosphatase.
In some embodiments, the pyrophosphatase is hot start.
In some embodiments, each well of the fourth region may further comprise one or more oligonucleotide binding dyes or molecular probes.
In some embodiments, each well of the third region may comprise at least one or more different A0 that is selective for a target sequence including an identification region.
In some embodiments, the amplification enzyme in the fourth region and the pyrophosphorolysing enzyme in the third region may be the same, thus in some embodiments the amplification enzyme in the fourth region is not needed.
In some embodiments, there may be a fifth region comprising one or more wells, wherein each well may comprise a proteinase and wherein said fifth region may be located between the first and second regions.
In some embodiments, the fifth region may be located between the second and third regions.
In some embodiments, the third and fourth regions of the device may be combined such that the wells of the third region further comprise:
In some embodiments, the means for detecting a signal are located within the third region.
In some embodiments, the means for detecting a signal are located within an adjacent region.
In some embodiments, the wells of the third or fourth region may further comprise:
In some embodiments, the amplification enzyme and the pyrophosphorolysis enzyme of the device are the same.
In some embodiments, the wells of the third region may comprise:
In some embodiments, the wells of the third region may further comprise:
In some embodiments, the wells of the third region may further comprise a plurality of HO1 and HO2.
In some embodiments, the wells of the third region may further comprise:
In some embodiments, the wells of the third region may comprise a partially double-stranded nucleic acid construct wherein:
In some embodiments, the wells of the third region may further comprise an enzyme for the removal of the at least one RNA base.
In some embodiments, the enzyme is Uracil-DNA Glycosylase (UDG) and the RNA base is uracil.
In some embodiments, one or more wells of the third region may further comprise:
In some embodiments, the double strand specific DNA digestion enzyme is an exonuclease.
In some embodiments, the double-strand specific DNA digestion enzyme is a polymerase with proofreading activity.
In some embodiments, the fluorophore is selected from dyes of the fluorescein family, the carboxyrhodamine family, the cyanine family, the rhodamine family, polyhalofluorescein-family dyes, hexachlorofluorescein-family dyes, coumarin-family dyes, oxazine-family dyes, thiazine-family dyes, squaraine-family dyes, and chelated lanthanide-family dyes.
In some embodiments, the fluorophore of the device may be selected from any of the commercially available dyes.
In some embodiments, the quencher of the device is selected from those available under the trade designations Black Hole™, Eclipse™ Dark, Qx1J, Iowa Black™, ZEN and/or TAO.
In some embodiments, the quencher of the device may be selected from any of the commercially available quencher.
In some embodiments, the wells of the third region may comprise one or more partially double stranded DNA constructs wherein each construct contains one or more fluorophores and one or more quenchers. In some embodiments, when construct is partially double-stranded the one or more fluorophores and one or more quenchers are located in close enough proximity to each other such that sufficient quenching of the one or more fluorophores occurs.
In some embodiments, the construct is one strand of DNA with a self-complementary region that is looped back on itself.
In some embodiments, the construct comprises one primer of a primer pair.
In some embodiments, the wells of the third region may further comprise the other primer of a primer pair.
In some embodiments, a portion of the single stranded section of the construct hybridises to A2 and is extended against it by a DNA polymerase. In some embodiments, the other primer of the primer pair then hybridises to the extended construct. This primer is then extended against the construct, displacing the self-complementary region. Thus, the one or more fluorophores and one or more dyes are separated sufficiently for a fluorescent signal to be detected, indicating the presence of A2 in the reaction mixture.
In such an embodiment the construct may be known as a Sunrise Primer.
In some embodiments, the construct comprises two separate DNA strands.
In some embodiments, a portion of the single stranded section of the construct hybridises to A2 and is extended against it by a DNA polymerase. In some embodiments, the other primer of the primer pair then hybridises to the extended construct, displaying A2. This primer is then extended against the construct, in the direction of the double stranded section, displacing the shorter of the DNA strands and thus the one or more fluorophores and one or more dyes are separated sufficiently for a fluorescent signal to be detected, indicating the presence of A2 in the reaction mixture.
In such an embodiment the construct may be known as a Molecular Zipper.
The person skilled in the art will appreciate that for both the Sunrise Primer and Molecular Zipper it is possible for the one or more fluorophores and the one or more quencher pairs to be located at various positions within each respective construct. The key feature is that each pair is located in sufficient proximity to one another that in the absence of A2, i.e. when no extension and strand displacement has occurred, no fluorescent signal is emitted.
In some embodiments, one or more wells of one or more regions may further comprise a pyrophosphatase.
In some embodiments, one or more wells of one or more regions of the device may further comprise a phosphatase or a phosphohydrolase.
In some embodiments, one or more wells of the second region of the device may further comprise an enzyme for the transcription of RNA into DNA.
In some embodiments, the enzyme is a reverse transcriptase.
In some embodiments, one or more enzymes present in the device are hot start.
In some embodiments, one or more enzymes present in the device are thermostable.
In some embodiments, the second and third regions of the device are combined.
In some embodiments, the third and fourth regions of the device are combined.
In some embodiments, there is located, between one or more wells of a region/and or between one or more regions of the device, one or more fluidic pathways.
In some embodiments, one or more of the first, second, third, fourth or fifth region may be fluidically connected to a sample container via a fluidic interface.
In some embodiments, heating and/or cooling elements may be present at one or more regions of the device.
In some embodiments, heating and/or cooling may be applied to one or more regions of the device.
In some embodiments, each region of the device may independently comprise at least 100 or 200 wells.
In some embodiments, each region of the device may independently comprise between about 100 and 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500 or more wells. The wells may be of any shape and their locations may be arranged in any format or pattern on a substrate.
In some embodiments, the well-substrate can be constructed from a metal (e.g. gold, platinum, or nickel alloy as non-limiting examples), ceramic, glass, or other PCR compatible polymer material, or a composite material. The well-substrate includes a plurality of wells.
In some embodiments, the wells may be formed in a well-substrate as blind-holes or through-holes. The wells may be created within a well-substrate, for example, by laser drilling (e.g. excimer or solid-state laser), ultrasonic embossing, hot embossing lithography, electroforming a nickel mold, injection molding, and injection compression molding.
In some embodiments, individual well volume may range from 0.1 to 1500 nl. In one embodiment, 0.5 to 50 nL. Each well may have a volume of about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, or 500 nL.
In some embodiments, well dimensions may have any shape, for example, circular, elliptical, square, rectangular, ovoid, hexagonal, octagonal, conical, and other shapes well known to persons of skill in the art.
In some embodiments, well shapes may have cross-sectional areas that vary along an axis. For example, a square hole may taper from a first size to a second size that is a fraction of the first size.
In some embodiments, well dimensions may be square with diameters and depths being approximately equal.
In some embodiments, walls that define the wells may be non-parallel.
In some embodiments, walls that define the wells may converge to a point. Well dimensions can be derived from the total volume capacity of the well-substrate.
In some embodiments, well depths may range from 25 μm to 1000 μm.
In some embodiments, wells may have a depth of 25, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950 or 1000 m.
In some embodiments, well diameter may range from about 25 μm to about 500 μm.
In some embodiments, wells may have a width of 25, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475 or 500 μm.
In some embodiments, portions of one or more regions of the device may be modified to encourage or discourage fluid adhered. Surfaces defining the wells may be coated with a hydrophilic material (or modified to be hydrophilic), and thus encourage retention of fluid.
In some embodiments, portions of one or more regions of the device, may be coated with a hydrophobic material (or modified to be hydrophobic) and thus discourage retention of fluid thereon. The person skilled in the art will understand that other surface treatments may be performed such that fluid is preferably held within the wells, but not on upper surfaces so as to encourage draining of excess fluid.
In some embodiments, the wells of the well-substrate may be patterned to have a simple geometric pattern of aligned rows and columns, or patterns arranged diagonally or hexagonally.
In one embodiment, the wells of the well-substrate may be patterned to have complex geometric patterns, such as chaotic patterns or isogeometric design patterns.
In some embodiments, the wells may be geometrically separated from one another and/or feature large depth to width ratios to help prevent cross-contamination of reagents.
In some embodiments, the device may comprise one or more auxiliary regions which are usable to provide process fluids, such as oil or other chemical solutions to one or more of the regions of the device. Such auxiliary regions may be fluidically connected to one or more of the regions of the device via one or more membranes, valves and/or pressure severable substrates (i.e. materials that break when subjected to a pre-determined amount of pressure from fluid within an auxiliary region or adjacent portion of the fluid pathway) such as metal foil or thin film.
In some embodiments, the fluid pathway of the device may include extensive torturous portions. A torturous path between the inlet passage of the fluid pathway and one or more of the regions of the device can be helpful for control and handling of fluid processes. A torturous path can help reduce formation of gas bubbles that can interfere with flowing oil through the fluid pathway.
In some embodiments, the device may further comprises a gas permeable membrane which enables gas to be evacuated from the wells of one or more regions of the device, while not allowing fluid to pass through. The gas permeable membrane may be adhered to the well-substrate of the device by a gas permeable adhesive. In one embodiment, the membrane may be constructed from polydimethylsiloxane (PDMS), and has a thickness ranging from 20-1000 μm. In some embodiments the membrane may have a thickness ranging from 100-200 μm.
In some embodiments, all or portions of the well-substrate may contain conductive metal portions (e.g., gold) to enable heat transfer from the metal to the wells. In one embodiment, the interior surfaces of wells may be coated with a metal to enable heat transfer.
In some embodiments, after appropriate reagents have filled the wells of one or more regions of the device, an isolation oil or thermally conductive liquid may be applied to the device to prevent cross-talk.
In some embodiments, the wells of one or more regions of the device may be shaped to taper from a large diameter to a smaller diameter, similar to a cone. Cone-shaped wells with sloped walls enables the use of a non-contact deposition method for reagents (e.g., ink jet). The conical shape also aids in drying and has been found to prevent bubbles and leaks when a gas permeable membrane is present.
In some embodiments, the wells of one or more regions of the device may be filled by advancing a sample fluid (e.g. via pressure) along the fluid pathway of the device. As the fluid passes over the wells of one or more regions of the device, each well becomes filled with fluid, which is primarily retained within the wells via surface tension. As previously described, portions of the well-substrate of the device may be coated with a hydrophilic/hydrophobic substance as desired to encourage complete and uniform filing of the wells as the sample fluid passes over.
In some embodiments, the wells of one or more regions of the device may be ‘capped’ with oil following filling. This can then aid in reducing evaporation when the well-substrate is subjected to heat cycling. In one embodiment, following oil capping, an aqueous solution can fill one or more regions of the device to improve thermal conductivity.
In some embodiments, the stationary aqueous solution may be pressurised within one or more regions of the device to halt the movement of fluid and any bubbles.
In some embodiments, oil such as mineral oil may be used for the isolation of the wells of one or more regions of the device and to provide thermal conductivity. However, any thermal conductive liquid, such as fluorinated liquids (e.g., 3M FC-40) can be used. References to oil in this disclosure should be understood to include such alternatives as the skilled person in the art will appreciate are applicable.
In some embodiments, the device may further comprise one or more sensor assemblies.
In some embodiments, the one or more sensor assemblies may comprise a charge coupled device (CCD)/complementary metal-oxide-semiconductor (CMOS) detector coupled to a fiber optic face plate (FOFP). A filter may be layered on top of the FOPF, and placed against or adjacent to the well-substrate. In one embodiment, the filter can be layered (bonded) directly on top of the CCD with the FOPF placed on top.
In some embodiments, a hydration fluid, such as distilled water, may be heated within the first region or one of the auxiliary regions such that one or more regions of the device has up to 100% humidity, or at least sufficient humidity to prevent over evaporation during thermal cycling.
In some embodiments, after filing of the device is complete, the well-substrate may be heated by an external device that is in thermal contact with the device to perform thermal cycling for PCR.
In some embodiments, non-contact methods of heating may be employed, such as RFID, Curie point, inductive or microwave heating. These and other non-contact methods of heating will be well known to the person skilled in the art. During thermal cycling, the device may be monitored for chemical reactions via the sensor arrangements previously described.
In some embodiments, reagents that are deposited in one or more of the wells of one or more of the regions of the device are deposited in a pre-determined arrangement.
In some embodiments there is provided a method comprising:
In some embodiments there is provided a method comprising:
In some embodiments of the method, the fluid pathway may be valve less.
In some embodiments of the method, the evacuated second region may be filled with a hydrophobic substance.
In some embodiments of the method, the evacuated third region may be filled with a hydrophobic substance.
In some embodiments of the method, the hydrophobic substance may be supplied from an oil chamber that is in fluid communication with the second and third regions.
In some embodiments of the method, the sample fluid may be routed along the fluid pathway in a serpentine manner.
In some embodiments, the method may further comprise applying heating and cooling cycles to the one or more of the first, second or third regions.
According to the present invention, there is provided an alternative method of detecting the methylation status of a target polynucleotide sequence in a given nucleic acid analyte characterised by the steps of:
In some embodiments, (a) comprises chemically or enzymatically converting the unmethylated cytosine bases in the target polynucleotide sequence.
In some embodiments, the converted polynucleotide target is introduced to a restriction endonuclease prior to or during step (b).
In some embodiments, the recognition sequence of the restriction endonuclease is created by the conversion performed in step (a).
In an alternative embodiment, the recognition sequence of the restriction endonuclease is created or removed by the conversion performed in step (a) and A0 is prevented from undergoing pyrophosphorolysis through chemical modification at or close to its 3′ end, or through a 3′ mismatch against the target sequence, and this modification or mismatch is removed through cleavage of A0 by the restriction endonuclease prior to pyrophosphorolysis.
In some embodiments, (a) comprises introducing the nucleic acid analyte to a methylation-sensitive or methylation-dependent restriction endonuclease.
In some embodiments, the restriction endonuclease employed cleaves copies of the polynucleotide target sequence in which the target state is not present.
In some embodiments, the restriction endonuclease and the first reaction mixture are added at the same time.
In some embodiments, A0 is prevented from undergoing pyrophosphorolysis through chemical modification at or close to its 3′ end, or through a 3′ mismatch against the target sequence, and this modification or mismatch is selectively removed when A0 is hybridised to a target molecule containing the epigenetic modification of interest through cleavage of A0 by the restriction endonuclease prior to pyrophosphorolysis.
In some embodiments, wherein (a) comprises introducing the nucleic acid analyte to a methylation-sensitive or methylation-dependent restriction endonuclease, (a) further comprises selective amplification of the target polynucleotide sequence containing the methylation status of interest through methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) of methylated DNA.
In some embodiments, the method comprises the method according to any previous embodiment wherein the products of (a) undergo PCR prior to (b).
In some embodiments, the method comprises the method according to any previous embodiment wherein the population of methylated or unmethylated target sequence is reduced prior to step (a).
In one embodiment, the reduction is carried out using methylated DNA immunoprecipitation (MeDIP).
In some embodiments, the reduction is carried out using methyl-binding proteins, such as MBD2b or the MBD2b/MBD3L1 complex.
The analytes to which the methods of the invention can be applied are as described previously.
In some embodiments, various versions of the method using different combinations of probe and trigger oligonucleotides (see below) are employed in parallel so that the analyte can be simultaneously screened for the detection of the methylation status of multiple target sequences; for example sources of cancer, cancer indicators or multiple sources of infection. In this approach, the amplified products obtained in step (e) by parallel application of the method are contacted with a detection panel comprised of one or more oligonucleotide binding dyes or sequence specific molecular probes such as a molecular beacon, hairpin probe or the like. Thus, in another aspect of the invention there is provided the use of at least one probe and optionally one trigger oligonucleotide defined below in combination with one or more chemical and biological probes selective for the target polynucleotide sequences or with the use of sequencing to identify the amplified probe regions.
Step (b) of the method of the invention comprises annealing the analyte whose presence in a given sample is being sought with a single-stranded probe oligonucleotide A0. In one embodiment this oligonucleotide comprises a priming region and a 3′ end which is complementary to the target polynucleotide sequence to be detected. By this means, a first intermediate product is created which is at least partially double-stranded. In one embodiment, this step is carried out in the presence of excess A0 and in an aqueous medium containing the analyte and any other nucleic acid molecules.
In some embodiments, where molecular probes are to be used for detection in step (e), the probe oligonucleotide A0 is configured to include an oligonucleotide identification region on the 5′ side of the region complementary to the target sequence, and the molecular probes employed are designed to anneal to this identification region. In one embodiment, only the complementary region of A0 is able to anneal to the target; i.e. any other regions lack sufficient complementarity with the analyte for a stable duplex to exist at the temperature at which step (c) is carried out. Here and throughout, by the term ‘sufficient complementarity’ is meant that, to the extent that a given region has complementarity with a given region on the analyte, the region of complementarity is more than 10 nucleotides long.
In one preferable embodiment, the 5′ end of A0 or an internal site on the 5′ side of the priming region is rendered resistant to exonucleolysis. By this means and after step (c), an exonuclease having 5′-3′ exonucleolytic activity can optionally be added to the reaction medium for the purpose of digesting any other nucleic acid molecules present whilst leaving A0 and any material comprising the partially digested strand A1 intact. Suitably, this resistance to exonucleolysis is achieved by introducing one or more blocking groups into the oligonucleotide A0 at the required point. In one embodiment, these blocking groups may be selected from phosphorothioate linkages and other backbone modifications commonly used in the art, C3 spacers, phosphate groups, modified bases and the like. In yet another, A0 has an oligonucleotide flap mismatch with respect to either or both of the 3′ and 5′ ends of the trigger oligonucleotide further described below.
In some embodiments, the identification region will comprise or have embedded within a barcoding region which has a unique sequence and is adapted to be indirectly identified in step (f) using a sequence-specific molecular probe applied to the amplified components A2 or directly by the sequencing of these components. Examples of molecular probes which may be used include, but are not limited to, molecular beacons, TaqMan® probes, Scorpion® probes and the like.
In step (c) of the method, the double-stranded region of the first intermediate product is pyrophosphorolysed in the 3′-5′ direction from the 3′ end of its A0 strand. As a consequence, the A0 strand is progressively digested to create a partially digested strand; hereinafter referred to as A1. Where the probe oligonucleotide erroneously hybridises with a non-target sequence, the pyrophosphorolysis reaction will stop at any mismatches, preventing subsequent steps of the method from proceeding. In another embodiment, this digestion continues until A1 lacks sufficient complementarity with the analyte or a target region therein to form a stable duplex. At this point, the various strands then separate by melting, thereby producing A1 in single-stranded form. Under typical pyrophosphorolysis conditions, this separation occurs when there are between 6 and 20 complementary nucleotides between the analyte and A0.
Suitably, pyrophosphorolysis carried out as described previously.
In one embodiment, step (c) is carried out in the presence of a phosphatase enzyme to continually remove by hydrolysis the nucleoside triphosphates produced by the pyrophosphorolysis reaction. In another embodiment, a pyrophosphatase enzyme is added after step (c) to hydrolyse any residual pyrophosphate ion thereby ensuring that no further pyrophosphorolysis can occur in later steps. In another embodiment, step (b) and (c) are iterated so that multiple copies of A1 are created from each target molecule. This may occur before or whilst the subsequent steps are being carried out. When combined with the amplification in step (e) this iteration leads to a further improvement in the sensitivity and reliability of the method and, by introducing an initial linear amplification, allows more accurate quantification of the target polynucleotide.
In one preferred, but non-essential, embodiment, at the end of step (c) or before or after step (d) an intermediate step is introduced in which an exonuclease having 5′-3′ directional activity is added for the purpose of ensuring that any residual nucleic acid material present, other than that comprised of the A0 or A1 strands (in which the 5′ blocking group is present), is destroyed. In another embodiment, this exonuclease is deactivated prior to step (e) being carried out. In yet another embodiment, prior to or whilst carrying out this exonucleolysis, all of the nucleic acid material present is phosphorylated at its 5′ ends using, for example a kinase and a phosphate donor such as ATP to produce a phosphorylated end site required for initiating the exonucleolysis by certain types of 5′-3′ exonucleases.
After step (c), or where relevant the intermediate step mentioned above, A1 is, in one embodiment (i), annealed to a single-stranded trigger oligonucleotide B to create a second intermediate product which is also partially double-stranded. In one embodiment, B is comprised of an oligonucleotide region complementary to the 3′ end of A1 with a flanking oligonucleotide region at its 5′ end which is not substantially complementary to A0. Here and throughout, by the term ‘not substantially complementary to’ or equivalent wording is meant that to the extent that a given flanking region has complementarity with a given region on A0, the region of complementarity is less than 10 nucleotides long. Thereafter, in step (d) the A1 strand of this second intermediate product is extended in the 5′-3′ direction to create a third intermediate product, comprised of B and extended A1 strand (hereinafter referred to as A2).
In another embodiment, B comprises (i) an oligonucleotide region complementary to the 3′ end of A1; (ii) an oligonucleotide region complementary to the 5′ end of A1 and optionally (iii) an intermediate oligonucleotide region between these two regions and wherein B is unable to undergo extension against A1 through the presence of either one or more nucleotide mismatches or a chemical modification at its 3′ end. In another embodiment, B is modified both at its 3′ end and internally to prevent other oligonucleotides being extended against it.
In both these embodiments, B is suitably comprised of oligonucleotide regions which are each independently up to 150 nucleotides, typically 5 to 100 nucleotides and most preferably 10 to 75 nucleotides long. In one embodiment, all the regions of B independently have a length in the range 10 to 50 nucleotides. In another preferred embodiment, the 5′ end of B or a region adjacent thereto is also protected with a blocking group of the type mentioned above to make it resistant to exonucleolysis. In some embodiments, the 5′ end of B is folded back on itself to create a double-stranded hairpin region. In yet another embodiment, both the 3′ and 5′ ends of B have one or more nucleotide mismatches with respect to the ends of its A1 counterpart strand.
In another embodiment, step (d) alternatively comprises (ii) ligating the two ends of A1 together in the presence of a ligase to create a third intermediate product in which the A1 strand is not extended but rather circularised. This ligation is typically carried out through the addition of a splint oligonucleotide D, having regions complementary to the 3′ and 5′ ends of A1 such that, when annealed to D, the 3′ and 5′ ends of A1 form a nick which can be ligated or a gap which can be filled prior to subsequent ligation. In this embodiment, circularised A1 in effect becomes A2 for the purpose of subsequent steps. In another embodiment, the A1 strand is still extended in the 5′-3′ direction, using a polymerase lacking in 5′-3′ exonuclease and strand-displacement activity, and is then circularised so that this extended and circularised product in effect becomes A2. In another embodiment, the 3′ and 5′ ends of A1, or extended A1 are joined together by a bridging-group which may not necessarily include an oligonucleotide region.
In the case where the third intermediate product comprises an A2 strand which is circularised, it is advantageous to treat the reaction mixture generated in step (d) with an exonuclease or combination of exonucleases to digest any residual nucleic acid components which are not circularised. Thereafter, in another embodiment the exonuclease is deactivated prior to step (e) taking place.
In another embodiment (iii), step (d) is carried out in the presence of a ligation probe C having a 5′ region complementary to at least part of a 5′ end region of a splint oligonucleotide D or to the target oligonucleotide, a ligase, and optionally a polymerase lacking both a strand displacement capability and 5′-3′ directional exonuclease activity. By such means, a second intermediate product is formed in which the A2 strand is comprised of A1, C and optionally an intermediate region formed by extension of A1 in the 5′-3′ direction to meet the 5′ end of C. In such an embodiment, the primers employed in step (e) (see below) are chosen to amplify at least a region of A2 including the site at which ligation of the A1 to C has occurred. In this embodiment, we have found that it is advantageous to include a 3′ blocking group on C so that a 3′-5′ exonuclease can be used to digest any non-ligated A1 prior to amplification. Suitable polymerases which may be used for the extension of A1 prior to ligation include but are not limited to Hemo KlenTaq, Mako and Stoffel Fragment.
In one embodiment, where steps d (ii) or d (iii) are employed A1 is optionally extended in 5′-3′ direction prior to ligation. In one embodiment this optional extension and the ligation are performed against the target oligonucleotide, while in another embodiment they are performed through addition of a further splint oligonucleotide D to which A1 anneals prior to extension and/or ligation. In one embodiment, D comprises an oligonucleotide region complementary to the 3′ end of A1 and a region complementary to either the 5′ end of oligonucleotide C or to the 5′ end of A1. In another embodiment, D is unable to extend against A1 by virtue of either a 3′-end modification or through a nucleotide mismatch between the 3′ end of D and the corresponding region of A1.
In subsequent step (e), the A2 strand or a desired region thereof is caused to undergo amplification so that multiple, typically many millions, of copies are made. This is achieved by priming a region of A2 and subsequently any amplicons derived therefrom with single-stranded primer oligonucleotides, provided for example in the form of a forward/reverse or sense/antisense pair, which can anneal to a complementary region thereon. The primed strand then becomes the point of origin for amplification. Amplification methods include, but are not limited to, thermal cycling and isothermal methods such as the polymerase chain reaction, recombinase polymerase amplification and rolling circle amplification; the last of these being applicable when A2 is circularised. By any of these means, many amplicon copies of A2 and in some instances its sequence complement can be rapidly created. The exact methodologies for performing any of these amplification methods will be well-known to one of ordinary skill and the exact conditions and temperature regimes employed are readily available in the general literature to which the reader is directed. Specifically, in the case of the polymerase chain reaction (PCR), the methodology generally comprises extending the primer oligonucleotide against the A2 strand in the 5′-3′ direction using a polymerase and a source of the various single nucleoside triphosphates until a complementary strand is produced; dehybridising the double-stranded product produced to regenerate the A2 strand and the complementary strand; re-priming the A2 strand and any of its amplicons and thereafter repeating these extension/dehybridisation/repriming steps multiple times to build-up a concentration of A2 amplicons to a level where they can be reliably detected.
Finally, in step, (f) the amplicons are detected and the information obtained used to infer whether the polynucleotide target sequence is present or absent in the original analyte and/or a property associated therewith. For example, by this means a target sequence characteristic of a cancerous tumour cell may be detected with reference to specific SNPs being looked for. In another embodiment, a target sequence characteristic of the genome of a virus of bacterium (including new mutations thereof) may be detected. Many methods of detecting the amplicons or identification regions can be employed including for example an oligonucleotide binding dye, a sequence-specific molecular probe such as fluorescently-labelled molecular beacon or hairpin probe. Alternatively, direct sequencing of the A2 amplicons can be performed using one of the direct sequencing methods employed or reported in the art. Where oligonucleotide binding dyes, fluorescently labelled beacons or probes are employed it is convenient to detect the amplicons using an arrangement comprising a source of stimulating electromagnetic radiation (laser, LED, lamp etc.) and a photodetector arranged to detect emitted fluorescent light and to generate therefrom a signal comprising a data stream which can be analysed by a microprocessor or a computer using specifically-designed algorithms.
In one specific manifestation of the invention, multiple A0 probes are employed each selective for a different target sequence and each including an identification region. In one embodiment, the region amplified in step (e) then includes this identification region. In another embodiment, the amplicons generated in step (e) are then inferred through detection of the identification region(s). Identification can then comprise using molecular probes or sequencing methods for example Sanger sequencing, Illumina® sequencing or one of the methods we have previously described. In another manifestation, prior to step (b) the analyte is split into multiple reaction volumes with each volume having a different probe oligonucleotide A0 or plurality thereof designed to detect different target sequence(s). In another preferred embodiment, the different probes A0 comprise a common priming site allowing a single or single set of primers to be used for amplification step (e).
In some embodiments, the amplification step (e) may be carried out by standard polymerase chain reaction (PCR) or through isothermal amplification such as rolling circle amplification (RCA). In some embodiments, the RCA may be in the form of exponential RCA for example hyperbranched RCA, which can result in double-stranded DNA of a variety of different lengths. In some embodiments, it may be desirable to provide different probes that can produce different products with different lengths.
In some embodiments, step (f) further comprises the steps of:
In some embodiments, step (f) may take the form of detection and analysis using melting curve analysis. Melting curve analysis can be an assessment of the dissociation characteristics of double-stranded DNA during heating. The temperature at which 50% of DNA in a sample is denatured into two separate stands is known as the melting temperature (Tm). As the temperature is raised, the double strand begins to dissociate, with different molecules of double-stranded DNA dissociating at different temperatures based on composition (a G-C base pairing has 3 hydrogen bonds compared to only 2 between A-T—thus a higher temperature is required to separate a G-C than an A-T), length (a longer length of double stranded DNA with more hydrogen bonds will require a higher temperature to fully dissociate into two separate single strands than one that is shorter) and complementarity (a DNA molecule with a large number of mismatches will have a lower Tm by nature of containing fewer hydrogen bonds between matching base pairs).
In some embodiments, the amplification step (e) may be carried out in the presence of an intercalating fluorescent agent. Thus, when the melting curve analysis is performed, changes in fluorescence are monitored, indicating the Tm (and so identity) of the reaction product and hence the target polynucleotide sequence. Changes in fluorescence can be detected using an arrangement comprising a source of stimulating electromagnetic radiation (laser, LED, lamp etc.) and a photodetector arranged to detect emitted fluorescent light and to generate therefrom a signal comprising a data stream which can be analysed by a microprocessor or a computer using specifically-designed algorithms.
The intercalating fluorescent agent may be dye specific to double-stranded DNA, such as SYBR Green®, EvaGreen, LG Green, LC Green Plus, ResoLight, Chromofy or SYTO 9. The person skilled in the art will appreciate there are many intercalating fluorescent agents which could be used in the current invention and the above list is not intended to limit the scope of the current invention. The intercalating fluorescent agent may be a fluorescently labelled DNA probe. In one embodiment of the invention, juxtapositioned probes, one comprising a fluorophore, the other a suitable quencher, can be used to determine the complementarity of the DNA probe to a target amplified sequence.
In one embodiment, the intercalating fluorescent agent may be Syto 82.
In another aspect of the invention, there is provided a method of detecting the epigenetic modification status of, and identifying, a target polynucleotide sequence in a given nucleic acid analyte characterised by the steps of:
In some embodiments, the denaturing conditions may be provided by varying the temperature e.g. increasing the temperature to a point where the double strand begins to dissociate. Additionally or alternatively, the denaturing conditions may also be provided by varying the pH such that the conditions are acidic or alkaline, or by adding in additives or agents such as a strong acid or base, a concentrated inorganic salt or organic solvent e.g. alcohol.
In a further aspect of the invention, the analyte in single-stranded form may be prepared from the biological sample mentioned above by a series of preliminary steps as already described previously.
In a further aspect of the invention there is provided an alternate embodiment in which the phosphorolysis step (c) is replaced with an exonuclease digestion step using a double-strand specific exonuclease. The person skilled in the art will understand that double-strand specific exonucleases include those that read in the 3′-5′ direction, such as ExoIII, and those that read in the 5′-3′ direction, such as Lambda Exo, amongst many others.
In one embodiment of this aspect, the double strand-specific exonuclease of step (c) proceeds in the 3′-5′ direction.
In some embodiments of this aspect, the double strand-specific exonuclease of step (c) proceeds in the 5′-3′ direction.
In embodiments of the invention wherein step (c) utilises a double strand-specific 5′-3′ exonuclease, it is the 5′ end of A0 that is complementary to the target analyte and the common priming sequence and blocking group are located on the 3′ side of the region complementary to the target. In a further embodiment, where molecular probes are to be used for detection in step (f), the probe oligonucleotide A0 is configured to include an oligonucleotide identification region on the 3′ side of the region complementary to the target sequence, and the molecular probes employed are designed to anneal to this identification region.
In embodiments of the invention wherein step (c) utilises a double strand-specific 5′-3′ exonuclease, an exonuclease having 3′ to 5′ exonucleolytic activity can optionally be added to the reaction mixture, after step (c) for the purpose of digesting any other nucleic acid molecules present whilst leaving A0 and any material comprising partially digested strand A1 intact. Suitably, this resistance to exonucleolysis is achieved as described previously.
It will be appreciated that the methods of the invention can be applied to a reaction mixture comprising a plurality of different analytes by using multiple different A0 and optionally B, C or D components, each associated with a different molecular probe or the like. In such a multiplexed method, the detection of multiple target regions characteristic of a given cancer or a multiplicity of infectious diseases etc. is enabled. In one embodiment, it is in preferred that every different A2 strand generated has a common primer site but different identification region, enabling one or a single set of primers to be used in amplification step (e).
In another aspect of the invention, there is provided the use of the methods described above to screen mammalian subjects, especially human patients, for the presence of infectious diseases, cancer or for the purpose of generating companion diagnostic information.
In a further aspect of the invention, there is provided control probes for use in the methods as described above. Embodiments of the current invention include those wherein the presence of a specific target sequence, or sequences, is elucidated by the generation of a fluorescent signal.
In such embodiments, there may inevitably be a level of signal generated from non-target DNA present in the sample. For a given sample, this background signal has a later onset than the ‘true’ signal, but this onset may vary between samples. Accurately detection of the presence of low concentrations of target sequence, or sequences, thus relies on knowledge of what signal is expected in its absence. For contrived samples references are available, but for true ‘blind’ samples from patients this is not the case. The control probes (E0) are utilised to determine the expected background signal profile for each assay probe. The control probe targets a sequence not expected to be present in the sample and the signal generated from this probe can then be used to infer the expected rate of signal generation from the sample in the absence of target sequence.
Thus there is provided a method of detecting the epigenetic modification status of a target polynucleotide sequence in a given nucleic acid analyte characterised by the steps of:
In some embodiments, the method in step (f) according to the present invention occurs by:
In some embodiments, the control probe (E0) and A0 are added to separate portions of the sample while in another embodiment the E0 and A0 are added to the same portion of the sample and different detection channels (e.g. different colour dyes) used to measure their respective signals. The signal generated by E0 may then be utilised to infer and correct for the background signal expected to be generated by A0 in the absence of the polynucleotide target sequence in the sample. For example, a correction of the background signal may involve the subtraction of the signal observed from E0 from that observed from A0, or through the calibration of the signal observed from A0 using a calibration curve of the relative signals generated by A0 and E0 under varying conditions.
In some embodiments, a single E0 can be used to calibrate all of the assay probes which may be produced.
In some embodiments, a separate E0 may be used to calibrate each amplicon of the sample DNA generated in an initial amplification step. Each amplicon may contain multiple mutations/target sequences of interest, but a single E0 will be sufficient to calibrate all of the assay probes against a single amplicon.
In a further embodiment, a separate E0 may be used for each target sequence. For example, if a C>T mutation is being targeted, an E0 could be designed that targets a C>G mutation in the same site that is not known to occur in patients. The signal profile generated by E0 under various conditions can be assessed in calibration reactions and these data used to infer the signal expected from the assay probe targeting the C>T variant when this variant is not present.
In some embodiments, of the method of the invention can be seen in
In step c, the partially double-stranded first intermediate product is pyrophosphorolysed with a pyrophosphorolysing enzyme in the 3′-5′ direction from the 3′ end of A0 to create a partially digested strand A1, the analyte and the undigested A0 molecule which did not anneal to a target in step a.
In
In step d, A2 is primed with at least one single-stranded primer oligonucleotide and multiple copies of A2, or a region of A2 are created.
In
In
In step d, A2 is primed with at least one single-stranded primer oligonucleotide and multiple copies of A2, or a region of A2 are created.
The specificity of the methods of the current invention may be improved by blocking at least a portion of wild-type DNA, promoting annealing of A0 only to the target polynucleotide sequences. Blocking oligonucleotides can be used to improve the specificity of the polymerase chain reaction (PCR). The general technique used is to design an oligonucleotide that anneals between the PCR primers and is not able to be displaced or digested by the PCR polymerase. The oligonucleotide is designed to anneal to the non-target (usually healthy) sequence, while being mismatched (often by a single base) to the target (mutant) sequence. This mismatch results in a different melting temperature against the two sequences, the oligonucleotide being designed to remain annealed to the non-target sequence at the PCR extension temperature while dissociating from the target sequence.
The blocking oligonucleotides may often have modifications to prevent its digestion by the exonuclease activity of the PCR polymerase, or to enhance the melting temperature differential between the target and non-target sequence.
The incorporation of a locked nucleic acid (LNA) or other melting temperature altering modification into a blocking oligonucleotide can significantly increase the differential in melting temperature of the oligonucleotide against target and non-target sequences.
Thus there is provided an embodiment of the invention wherein blocking oligonucleotides are used. The blocking oligonucleotides must be resistant to the pyrophosphorolysing (PPL) reaction to ensure they are not digested or displaced. This can be achieved in a number of different ways, for example via mismatches at their 3′ ends or through modifications such as phosphorothioate bonds or spacers.
In such embodiments or an aspect of the present invention where blocking oligonucleotides are used, the method of detecting a target polynucleotide sequence in a given nucleic acid analyte is characterised by the steps of:
In one embodiment, the blocking oligonucleotides are made to be resistant to the pyrophosphorolysing reaction via mismatches at their 3′ ends. In another embodiment, the blocking oligonucleotides are made to be resistant via the presence of a 3′-blocking group. In another embodiment the blocking oligonucleotides are made to be resistant via the presence of spacers or other internal modifications. In a further embodiment the blocking oligonucleotides include both a melting temperature increasing modification or modified nucleotide base and are rendered resistant to pyrophosphorolysis.
In one embodiment, A1 is circularised against the analyte target sequence. In this embodiment, the region of the target that is revealed by progressive digestion of A0, in the 3′-5′ direction from the 3′ end of A0 to form A1, is complementary to the 5′ end of A0/A1. In this embodiment, a ligase may be used to ligate the 3′ and 5′ ends of A1 to form a circularised oligonucleotide A2. This is shown, for example in
The invention is now illustrated with reference to the following experimental data.
A single-stranded first oligonucleotide 1 (SEQ ID NO 1) was prepared, having the following nucleotide sequence:
wherein A, C, G, and T represent nucleotides bearing the relevant characteristic nucleobase of DNA.
A set of single-stranded oligonucleotides 2-6 (SEQ ID NOs 2-6) was also prepared, having the following nucleotide sequences in the 5′ to 3′ direction:
wherein oligonucleotide 2 includes a 52 base region complementary to the 52 bases at the 3′ end of oligonucleotide 1 and oligonucleotides 3-6 include the same region with single nucleotide mismatches at positions 1, 10, 20 and 30 respectively.
A reaction mixture was then prepared, having a composition corresponding to that derived from the following formulation:
Pyrophosphorolysis of oligonucleotide 1 was then carried out by incubating the mixture at 37° C. for 120 minutes and the resulting reaction product analysed by gel electrophoresis.
The results of this analysis are shown in
Single-stranded first oligonucleotides 1 (SEQ ID NO 7) and 2 (SEQ ID NO 8) were prepared, having the following nucleotide sequences:
wherein A, C, G, and T represent nucleotides bearing the relevant characteristic nucleobase of DNA and P represents a 5′ phosphate group, and wherein oligonucleotide 1 comprises a shortened oligonucleotide 2 as would be obtained through pyrophosphorolysis of oligonucleotide 2 against a suitable target oligonucleotide.
A third single-stranded oligonucleotide 3 (SEQ ID NO 9) was also prepared, having the following nucleotide sequence:
wherein/3ddC/represents a 3′ dideoxycytosine nucleotide, and wherein oligonucleotide 3 has a 5′ end complementary to the 3′ end of oligonucleotide 1 and an internal region of oligonucleotide 2, and a 3′ end complementary to the 5′ ends of oligonucleotides 1 and 2.
A reaction mixture was then prepared, having a composition corresponding to that derived from the following formulation:
A second reaction mixture was then prepared, having a composition corresponding to that derived from the following formulation:
The first and second reaction mixes were then combined, and the resulting mix incubated at 37° C. for 30 minutes to allow exonucleolytic digestion of any uncircularised DNA. The resulting solution was then analysed by gel electrophoresis.
The results of this analysis are shown in
A pair of single stranded oligonucleotide primers 1 (SEQ ID NO 10) and 2 (SEQ ID NO 11) were prepared, having the following nucleotide sequences:
wherein A, C, G, and T represent nucleotides bearing the relevant characteristic nucleobase of DNA.
A reaction mixture was then prepared, having a composition corresponding to that derived from the following formulation:
The second reaction mix was then combined with 0.1 uL of the first reaction mix, and the resulting mixture incubated at 98° C. for 1 minute followed by 30 cycles of (98° C.×20 sec; 55° C.×30 sec; 68° C.×30 sec) to allow exponential amplification to take place via the polymerase chain reaction.
The resulting reaction product was then analysed by gel electrophoresis, the results of which are shown in
A single-stranded first oligonucleotide 1 (SEQ ID NO 12) was prepared, having the following nucleotide sequence:
wherein A, C, G, and T represent nucleotides bearing the relevant characteristic nucleobase of DNA; F represents a deoxythymidine nucleotide (T) labelled with Atto 594 dye using conventional amine-attachment chemistry and Q represents a deoxythymidine nucleotide labelled with a BHQ-2 quencher.
Another single-stranded oligonucleotide 2 (SEQ ID NO 13) was also prepared, having the following nucleotide sequence:
wherein X represents an inverted 3′ dT nucleotide, such that when oligonucleotide 2 is annealed to oligonucleotide 1 the 3′ end of oligonucleotide 1 is recessed, making it a target for pyrophosphorolysis, while the 3′ end of oligonucleotide 2 is protected from pyrophosphorolysis by the presence of the terminal inverted nucleotide.
A reaction mixture was then prepared, having a composition corresponding to that derived from the following formulation:
Pyrophosphorolysis of oligonucleotide 1 was then carried out by incubating the mixture at 37° C. for 75 minutes. As oligonucleotide 1 was progressively pyrophosphorolysed, the fluorescent dye molecules were separated from the quenchers and were then able to generate a fluorescent signal. The growth in this fluorescence during the incubation was monitored using a CLARIOStar microplate reader (ex. BMG Labtech) and used to infer the rate of pyrophosphorolysis of the oligonucleotide in the presence of inorganic pyrophosphate, imidodiphosphate or water. The results of this experiment are shown graphically in
The methods of the current invention were performed to detect the presence of, and identify, three different mutations which can occur in the human EGFR gene: T790M (exon 20), C797S (exon 20) and L861Q (exon 21).
Six samples containing wild-type genomic DNA were prepared. Three of these samples were spiked with a single synthetic mutant sequence for each of the three mutations of interest such that the final mutant allele fraction in these samples was 1%. To each sample was added a probe oligo A0 designed for the detection of a different single mutation:
The samples were subjected to pyrophosphorolysis through addition of inorganic pyrophosphate ion and Mako DNA polymerase and incubation at 41° C. Following pyrophosphorolysis of the probe oligos, ligation was performed through addition of E. coli Ligase and splint oligos with the following sequences:
Following ligation, the samples were subjected to hyperbranched rolling circle amplification through addition of dNTPs, BstLF DNA polymerase, Sybr Green® intercalating dye, a mutation-specific forward primer and a universal reverse primer having the sequences below, followed by incubation at 60° C. for 70 minutes.
The temperature of the samples was then increased from 70° C. to 95° C. and a fluorescence measurement taken at every 0.5° C. The resulting data curves were differentiated to produce melting peaks, the results of which are shown in
The following applications described below provide some examples of how the methods of the present invention can be applied.
The methods of the present invention can be used to detect specific genetic markers in a sample which may be used to help guide the selection of appropriate therapy. These markers may be tumour-specific mutations, or may be wild-type genomic sequences, and may be detected using tissue, blood or any other patient sample type. The markers may be epigenetic markers.
Repeated testing of patient samples during treatment of disease may allow early detection of developed resistance to therapy. As an example of this application is in non-small cell lung carcinoma (NSCLC), in which epidermal growth factor receptor (EGFR) inhibitors (e.g. gefitinib, erlotinib) are commonly used as first line treatments. During treatment the tumour can often develop mutations in the EGFR gene (e.g. T790M, C797S) which confer resistance to the drug. Early detection of these mutations may allow for transfer of the patient onto alternative therapies (e.g. Tagrisso). Epigenetic changes to the DNA of a patient can indicate the development of resistance.
Typically patients being monitored for resistance onset can be too sick for repeated tissue biopsy to be carried out. Repeated tissue biopsy may also be expensive, invasive and carries associated risks. It is preferable to test from blood, but there may be very low copy numbers of the mutations of interest in a reasonable blood drawn sample. Monitoring therefore requires sensitive testing from blood samples using a method of the present invention in which the method is simple and cost effective to carry out such that it can be regularly performed.
In this application example, patients who have been declared free of disease following treatment may be monitored over time to detect the recurrence of disease. This needs to be done non-invasively and requires sensitive detection of target sequences from blood samples. By using the method of the present invention, it provides a simple and low-cost method that can be regularly performed. The sequences targeted may be generic mutations known to be common in the disease of interest, or can be custom panels of targets designed for a specific patient based on detection of variants in the tumour tissue prior to remission.
For some cancers there are residual cancer cells that remain in a patient after treatment, it is a major cause of relapse in cancer and leukaemia. MRD monitoring and testing has several important roles: determining whether treatment has eradicated the cancer or whether traces remain, comparing the efficacy of different treatments, monitoring patient remission status as well as detecting recurrence of leukaemia, and choosing the treatment that will best meet those needs.
Population screening for early detection of disease has been a long-held goal, particularly in cancer diagnostics. The challenge is two-fold: the identification of panels of markers which allow confident detection of disease without too many false negatives, and the development of a method with sufficient sensitivity and low enough cost. The methods of the present invention could be used to address larger panels of mutations than PCR-based tests but with a much simpler workflow and lower cost than sequencing-based diagnostics.
When a transplanted organ is rejected by the recipient, the DNA from this organ is shed into the recipient's bloodstream. Early detection of this DNA would allow early detection of rejection. This could be achieved using custom panels of donor-specific markers, or by using panels of variants known to be common in the population, some of which will be present in the donor and some in the recipient. Routine monitoring of organ recipients over time could be enabled by the low cost and simple workflow of the present invention disclosed herein.
It has long been known that fetal DNA is present in maternal blood, and the NIPT market is now quite saturated with companies using sequencing the identify mutations and count copy numbers of specific chromosomes to enable detection of fetal abnormalities. The methods of the present invention as disclosed herein have the ability to detect mutations at very low allele fractions, potentially allowing earlier detection of fetal DNA. Identification of common mutations in a given population would allow assays to be developed that target mutations that may be present in either the maternal or fetal DNA or to allow detection of abnormalities at an earlier stage of pregnancy.
In some instances, there are groups of mutations or target sequences for which the presence but not the identity of any one of the targets should be identified. In others, information is required on both the presence and the identity of the mutation or sequences. In both instances, it is beneficial to multiplex the reaction such that multiple targets are assayed in a single reaction volume. This leads to improved efficiency of the process, increasing either the number of samples that can be processed at one time or the size of the panel of targets that can be assayed. When the presence but not the identity of a target sequence is required, multiplexing can be as simple as combining the probes for multiple targets into a single reaction volume. One key advantage of the methods of the current invention over standard PCR is that one single set of primers can be used to amplify all of the ‘activated’ probes (A2) in the final step of the reaction.
Using Exon19 deletions on the EGFR gene, the inventors have demonstrated 10-fold multiplexed detection at 0.1% mutant allele frequency (MAF) in a single reaction.
20 samples were prepared, each comprising wildtype (WT) DNA spiked with 0.1% or 0.5% of one of 10 different Exon19 deletions. An addition sample comprising only WT DNA was used as a control. Probes for the detection of all 10 different Exon19 mutations were added to every sample and the reaction performed using standard conditions. The results (see
Detection can be performed using standard techniques—intercalating dye, labelled probes (Taqman, Scorpion, stem-loop primers), molecular beacons or any of the other standard techniques which will be known to the person skilled in the art.
When the identity of the target is required it is most likely that a multi-colour system is used to identify which probe has been activated (A2). This naturally necessitates a probe design in which there are different ‘barcode’ sequences in the probes for different targets, which are then used for identification. Identification can then be performed as discussed previously.
As well as 10-plex multiplexed detection using 1 colour, the inventors have also demonstrated two-colour detection in a single well in both the linear and rolling-circle amplification implementations of the current methods (the former using Taqman probes, the latter using stem-loop primers). In this example, samples were prepared containing either the T790M mutation or the C797S mutation at allele fractions of 0%, 0.1% and 0.5%. Following pyrophosphorolysis and subsequent ligation, the samples were subjected to rolling circle or linear PCR amplification using primers or probes specific to the mutation-targeting probes A0 labelled with different fluorophores. The results of the rolling circle amplification using labelled stem-loop primers are shown in
Three samples 1-3 were prepared, each comprising 100 nM final concentration of a synthetic oligonucleotide 1 (SEQ ID NO 24) comprising the wild-type sequence of the L858R mutation region of exon 21 of the human EGFR gene:
A synthetic ‘mutant’ oligonucleotide 2 (SEQ ID NO 25) was prepared, having the following sequence derived from the same region of the EGFR gene and further comprising the L858R mutation:
Oligonucleotide 2 was added to samples 2 and 3 at 100 pM and 1 nM final concentrations respectively such that 0.1% of the molecules comprising the L858R mutation site in sample 2 and 1% of those in sample 3 included this mutation. Each sample was then split into two reaction volumes. To the first reaction volume an assay probe oligonucleotide 3 (SEQ ID NO 26) was added at 10 nM final concentration which comprised a 3′ end perfectly matching the mutated L858R sequence region while to the second volume a control probe oligonucleotide 4 (SEQ ID NO 27) was added at the same concentration which comprised the same sequence other than in the L858R mutation region in which it comprised a sequence mismatched to both the mutant and wild-type alleles:
The reaction volumes were then subjected to pyrophosphorolysis through the addition of 0.6 mM pyrophosphate ion and 37.5 U/mL Mako DNA polymerase and heating to 41° C. for 30 minutes. Following this reaction a splint oligonucleotide 5 (SEQ ID NO 28) was added to each reaction volume at 10 nM final concentration along with 50 U/mL Thermostable Inorganic Pyrophosphatase and 100 U/mL E. coli Ligase and any pyrophosphorolysed probes were circularised through incubation at 37° C. for 10 minutes. The E. coli Ligase was then inactivated through heating to 95° C. for 10 minutes.
Following this, the samples were subjected to exonuclease digestion through addition of Exonuclease III and T5 Exonuclease and incubation at 30° C. for 5 minutes followed by inactivation of the exonucleases through heating to 95° C. for 5 minutes.
To each sample was then added two primer oligonucleotides 6 (SEQ ID NO 29) and 7 (SEQ ID NO 30) at 200 nm final concentration, 0.4 mM dNTPs, 320 U/mL BstLF DNA polymerase and 0.5× final concentration Sybr Green® intercalating dye.
The samples were incubated at 60° C. for 80 minutes and the fluorescence from the Sybr Green® dye in each sample measured once per minute. The results of this incubation are shown in
For the purpose of this, and following sections, embodiments of the invention are exemplified and referred to as Protocol 1-5 respectively.
The table below shows an overview of the time taken to perform each Protocol:
In one embodiment, TIPP is absent from any one of the protocols, methods, kits and or devices of the invention.
In one embodiment, a 5′-3′ exonuclease is absent from any one of the protocols, methods, kits and or devices of the invention.
The tables below show an overview of the enzymes used in each Protocol:
In one embodiment, the presence of pyrophosphatase is optional.
In one embodiment, the presence of a 5′-3′ exonuclease is optional.
In one embodiment, the presence of UDG is optional.
As can be seen, the inventors have reduced total number of enzymes needed thus reducing the cost and complexity of the method. Surprisingly, the inventors discovered that moving the 5′-3′ exonuclease addition from the pre-amplification step to the pyrophosphorolysis/ligation step of the protocol (as in protocols 3-5) results in a higher fluorescent signal (representing detection of particular target analyte sequence) as shown in
The inventors have tested the method of Protocol 3 of the current invention using a range of different PPL enzymes, the results of which can be seen in
The inventors have tested the method of Protocol 4 of the current invention using a range of different PPL enzymes, the results of which can be seen in
The inventors have detected Exon19 del_6223 at 0.5%, 0.10% and 0.05% MAF, which can be seen in
The inventors have detected EGFR exon 20 T790M mutations at 0.10%, 0.50% and 1% MAF as shown in
The inventors have demonstrated that an exonuclease digestion step during RCA is not essential. However, a detectable signal is detected later in the RCA if the exonuclease digestion step is omitted.
The inventors have investigated what effect the PPL:RCA mix ratio has on the intensity of signal detected for 0.5% MAF EGFR exon 20 T790M, the results of which are shown in
The inventors have investigated whether the dye used during RCA can be optimised.
A lower RCA temperature may allow the methods of the invention to be carried out in a plate reader instead of qPCR.
The reaction utilising Syto82 is faster, as can be seen in
The inventors have investigated the use of two different enzymes, BST L.F and BST 2.0 WS, for RCA according to Protocol 4 to detect 0.5% MAF EGFR exon 20 T790M mutation. The results of this are shown in
The inventors have investigated the effect of different PPL enzymes on the RCA reaction at different PPL:RCA reaction mixture ratios. The results of which can be seen in
A single-stranded first oligonucleotide 1 (SEQ ID NO 31) was prepared, having the following nucleotide sequence:
A single-stranded ligating oligonucleotide 2 (SEQ ID NO 32) was prepared, having the following nucleotide sequence:
wherein A, C, G, and T represent nucleotides bearing the relevant characteristic nucleobase of DNA,
/5Phos/represent 5′ end phosphate
* represent phosphorothioate bond
A set of single-stranded oligonucleotides 3-4 (SEQ ID NOs 33-34) was also prepared, having the following nucleotide sequences in the 5′ to 3′ direction:
wherein oligonucleotide 3 includes a 17 base region complementary to the 17 bases at the 3′ end of oligonucleotide 1 and oligonucleotide 4 include the same region with single nucleotide mismatches at positions 3.
A first reaction mixture was then prepared, having a composition corresponding to that derived from the following formulation:
Pyrophosphorolysis, followed by circularisation, via ligation of, oligonucleotide 1 was then carried out by incubating the mixture at 45° C. for 15 minutes and the resulting product mixture was used in the amplification reaction (Example 18).
A pair of single stranded oligonucleotide primers 1 (SEQ ID NO 35) and 2 (SEQ ID NO 36) were prepared, having the following nucleotide sequences:
wherein A, C, G, and T represent nucleotides bearing the relevant characteristic nucleobase of DNA.
A second reaction mixture was then prepared, having a composition corresponding to that derived from the following formulation:
The reaction mix was then incubated at 50° C. for 40 minute and the resulting reaction product was then analysed by real-time fluorescence. The results of which are shown in
WT oligo dilution is made up of the following ingredients:
T790M and C797S 1% AF mutant oligo mix:
The PhusionU buffer composition is not publicly available.
A mixture was prepared corresponding to:
A mixture was prepared corresponding to:
A mixture was prepared corresponding to:
This mixture was then incubated at 37° C. for 10 min, 95° C. for 10 min.
A mixture was prepared corresponding to:
This mixture was then incubated at 30° C. for 5 min, 95° C. for 5 min.
A mixture corresponding to the following was prepared:
The mixture was then incubated at 60° C. for 90 min. Fluorescent measurements were taken every 1 minute. Cq was obtained based on auto-threshold given by Bio-rad machine. The results of this can be seen in
1. Target oligo dilution
G719X_6239, G719X_6252, G719X_6253 0.5% AF mutant oligonucleotide mix:
The Q5 U buffer composition is not publicly available.
A mixture was prepared corresponding to:
This mixture was then incubated at 45° C. for 15 min.
A mixture corresponding to the following was prepared:
Dye primer mix 1 consists of:
The mixture was then incubated at 58° C. for 150 min. Fluorescent measurements were taken every 1 minute. The results of this can be seen in
1. Oligonucleotide dilution
A methylated mix was created with following mixture of oligonucleotides (Mix1) diluted in H2O:
An unmethylated mix was created with following mixture of oligonucleotides (Mix2) diluted in H2O:
For both mixes the concentration of oligonucleotides was measured using Qubit™ 4 Fluorometer (ThermoFisher cat. Q33238) and Qubit™ dsDNA HS Assay Kit (ThermoFisher cat. Q32851) following the manufacturer protocol.
For strand conversion, two commercially available kits wereused:
Methylated and unmethylated strands were mixed together to create mixtures comprising different perchance methylated vs un methylated strands (1.56%-100%).
A PPL mixture was prepared corresponding to:
This mixture was then incubated at 45° C. for 15 min.
A mixture corresponding to the following was prepared:
The mixture was then incubated at 62° C. for 90 min. Fluorescent measurements were taken every 1 minute. The results of this can be seen in
Oligo solutions were prepared as following
A PPL mixture was prepared corresponding to:
This mixture was then incubated at 37° C. for 45 min.
A mixture corresponding to the following was prepared:
The mixture was then incubated at 62° C. for 60 min. Fluorescent measurements were taken every 1 minute. The results of this can be seen in
Dilution of oligonucleotides were prepared in 0.5×A7 and 0.5×Q5 buffer:
A PPL mixture was prepared consisting of:
The Q5 buffer composition is not publicly available.
The resulting mixture was incubated at 45° C. for 15 minutes.
A RCA mixture was prepared consisting of:
The resulting mixture was incubated at 50° C. for 70 minutes.
Fluorescent readings were taken every 1 minute. The results can be seen in
Lung cancer is a leading cause of cancer-associated mortality for a number of reasons, including its late manifestation of symptoms and the low sensitivity of screening techniques such as chest radiography. DNA fragments shed from tumour cells can provide a convenient and minimally invasive access to the molecular portrait of cancer, with these DNA fragments being found in the cell free DNA (cfDNA) isolated from the blood of cancer patients. Cell-free circulating tumour DNA (ctDNA) in the blood plasma presents a surrogate for the entire cancer genome; ctDNA accounts for as low as 0.05% of total cfDNA or less in many cancer patients, especially in the early stages of the disease. These properties make aberrant methylation of ctDNA a promising cancer biomarker, and recent high throughput investigations have demonstrated the correspondence between the changes in methylation profiles of ctDNA and DNA from paired tumour tissue. A list of methylated markers for lung cancer diagnostics and prognosis are shown below in:
The O6-methylguanine-DNA methyltransferase (MGMT) gene encodes an evolutionarily conserved and ubiquitously expressed methyltransferase involved in DNA repair. MGMT removes alkyl adducts from the 06-position of guanine, preventing DNA damage and imparting a protective effect on normal cells. However, the endogenous function of MGMT also protects tumour cells from the otherwise lethal effects of chemotherapy with alkylating agents such as temozolomide (TMZ). Silencing or reduced expression of MGMT through methylation of its respective gene promoter has been observed in 50% of grade IV gliomas, compromising DNA repair and as a result increasing chemosensitivity to agents such as TMZ. Therefore, MGMT promoter methylation status has potential as a biomarker of sensitivity to alkylating chemotherapy, ultimately influencing clinical practice. Its capacity as both a predictive and prognostic biomarker has been studied extensively, however, at present there is no consensus on the optimal method of assessment of MGMT gene promoter methylation.
Telomere maintenance protects the integrity of chromosomal ends, enabling replicative immortality, a hallmark of human cancer. The telomere reverse transcriptase (TERT) oncogene encodes the rate-limiting catalytic subunit of the telomerase holoenzyme, which is responsible for telomere maintenance and is normally only expressed in a subset of stem cells. The TERT gene is reactivated in approximately 90% of cancer cells, allowing indefinite proliferation and immortalisation of these cell types. A variety of genetic and epigenetic mechanisms underlying TERT dysregulation have been identified, with hypermethylation of the TERT promoter region representing a unique characteristic of cancer cells. Interestingly, methylation, and not mutation, in the upstream of transcription start site (UTSS) of the TERT gene was found to be strongly associated with increased TERT expression and poor prognosis in paediatric brain tumours. Given the prevalence of TERT promoter hypermethylation in a wide variety of cancer cell types, this epigenetic modification represents a useful prognostic biomarker.
Methylation of Prostate Cancer Genes Etc. Which Genes are/are not Methylated
Prostate cancer is the most frequently diagnosed non-skin malignancy, and a leading cause of cancer-related death in men in Western industrialised countries. There are many DNA methylation changes observed between benign and cancerous prostate tissues, with changes frequently being early and recurrent, suggesting a potential functional role. Several genes and gene families have been reported to be recurrently hypermethylated in prostate cancer by multiple genome-wide studies. This includes, but is not limited to, GSTP1, MGMT, AR, ER, VHL, RB1, APC, DAPK, CD44, AOX1, APC, CDKN2A, HOXD3, PTGS2, RARB, WT1, ZNF154, C200rf103, EFS, HOXC11, LHX9, RUNX3, TBX15, BARHL2, BDNF, CCDC8, CYP27A1, DLX1, EN2, ESR1, FBLN1, FOXE3, GP5, FRSP, HHEX, HOXA3, HOXD4, HOXD8. IRX1, KIT, LBX1, LHX2, NKX2-1, NKX2-2, NKX2-5, PHOXRA, POU3F3, RHCG, SIX6, TBX3, TMEM106, VAX1, and WNT2.
Pancreatic ductal adenocarcinoma (PDAC) is one of the most deadly cancer types. This form of cancer is difficult to diagnose as there are currently no early diagnostic tests available, meaning diagnosis usually occurs when the disease is already in an advanced state (>75% of diagnosed cases are stage III/IV diseases). This has led to high mortality rates being recorded. Early diagnosis has proven difficult due to the lack of reliable biomarkers able to capture the early development and/or progression of PDAC. Currently, the only FDA approved biomarker for prognostic surveillance of patients with PDAC is carbohydrate antigen 19-9 (CA19-9 or sialylated Lewis antigen). This antigen displays low sensitivity and specificity for the detection of disease. Its use is therefore discouraged for diagnostic purposes unless used in combination with other circulating biomarkers.
Recent studies have shown that cell-free DNA (cfDNA) methylation analysis represents a promising non-invasive approach for the discovery of biomarkers with diagnostic potential. It may be possible for cfDNA methylation to be utilised for the identification of disease-specific signatures in pre-neoplastic lesions or chronic pancreatitis (CP). As CP often precedes PDAC, dynamic DNA methylation patterns for a given set of genes may underlie the progression of the disease. CUX2, a ductal cell marker, has shown increased signal in PDAC; and REG1A, a ductal and acing cell marker, has shown increasing signal in chronic pancreatitis. Biomarkers ADAMTS1 and BNC1 have been seen to have high methylation frequency in primary PDACs and in pre-neoplastic pancreatic intra-epithelial neoplasia (PINs) (25% and 70% for ADAMTS1 and BNC1, respectively). The combined cfDNA methylation of ADAMTS1 and BNC1 may be utilised for the early diagnosis of pancreatic cancers (i.e. stages I and II). A list of potential biomarkers are shown in the below:
The KRAS gene controls cell proliferation, when it is mutated this negative signalling is disrupted and cells are able to continuously proliferate, often developing into cancer. A single amino acid substitution, and in particular a single nucleotide substitution, is responsible for an activating mutation implicated in various cancers: lung adenocarcinoma, mucinous adenoma, ductal carcinoma of the pancreas and colorectal cancer. KRAS mutations have been used as prognostic biomarkers, for example, in lung cancer.
Driver mutations in KRAS are associated with up to 20% of human cancers and there are targeted therapies in development against this mutation and its associated disease(s), a non-limiting list of some such therapies can be seen in the table below:
The presence of KRAS mutation has been found to reflect a very poor response to the EGFR inhibitors panitumumab (Vectibix) and cetuximab (Erbitux). Activating mutations in the gene that encodes KRAS occurs in 30%-50% of colorectal cancers and studies show that patients whose tumours express this mutated version of the KRAS gene will not respond to panitumumab and cetuximab. The presence of the wild-type KRAS gene is not a guarantee that a patient will respond to these drugs, however, studies have shown that cetuximab has significant efficacy in metastatic colorectal cancer patients with wild-type KRAS tumours. Lung cancer patients who are positive for KRAS mutation (wild-type EGFR) have a response rate estimated at 5% or less for the EGFR antagonists erlotinib or gefitinib compared with a 60% response rate in patients who do not possess a KRAS mutation.
The early detection of the emergence of KRAS mutations (activating or over-expression), a frequent driver of acquired resistance to cetuximab therapy (anti-EGFR therapy) in colorectal cancers, allows the modification of treatment (for example the early initiation of a mitogen-activated protein kinase kinase [MEK] inhibitor) to delay or reverse resistance and thus it is advantageous that the methods of the current invention allow the quick and cheap detection of the KRAS status of patients.
A non-limiting list of mutations is: G12D, G12A, G12C, G13D, G12V, G12S, G12R, A59T/E/G, Q61H, Q61K, Q61R/L, K117N and A146P/T/V.
A further non-limiting list of mutations is shown in the table below:
BRAF is a human gene that encodes for a protein called B-Raf which is involved in sending signals inside cells which are involved in directing cell growth. It has been shown to be mutated in some human cancers. B-Raf is a member of the Raf kinase family of growth signal transduction protein kinases and plays a role in regulating the MAP Kinase/ERKs signalling pathway, which affects, amongst other things, cell division.
Certain other inherited BRAF mutations cause birth defects.
More than 30 mutations of the BRAF gene have been identified that are associated with human cancers. In 90% of cases, thymine is substituted with adenine at nucleotide 1799. This leads to valine (V) being substituted for by glutamate (E) at codon 600 (now referred to as V600E) in the activation segment found in human cancers. This mutation has been widely observed in:
A non-limiting list of other mutations which have been found are: R4611, 1462S, G463E, G463V, G465A, G465E, G465V, G468A, G468E, N580S, E585K, D593V, F594L, G595R, L596V, T5981, V599D, V599E, V599K, V599R, V600K and A727V.
Drugs that treat cancers driven by BRAF mutations have been developed; vemurafenib and dabrafenib are approved by the FDA for the treatment of late-stage melanoma. The response rate to treatment with vumerafenib was 53% for metastatic melanoma, compared to 7-12% for the former best chemotherapeutic dacarbazine.
Human epidermal growth factor receptor 2 (HER2), also known as CD340 (cluster of differentiation 340), proto-oncogene Neu, Erbb2 (rodent) or ERBB2 (human) is a protein encoded by the ERBB2 gene. Amplification or over-expression of this oncogene plays an important role in the progression of aggressive types of breast cancer. Over-expression of the ERBB2 gene is also known to occur in ovarian, stomach, adenocarcinoma of the lung, and aggressive forms of uterine cancer and 30% of salivary duct carcinomas. Structural alterations have also been identified that cause ligand-independent firing of the receptor in the absence of over-expression.
There are numerous targeted therapies approved and in development against this mutation and its associated disease(s), a non-limiting list of some such therapies can be seen in the table below:
HER2 testing is routinely performed in breast cancer patients to assess prognosis, monitor response to treatment and to determine suitability for targeted therapy (trastuzumab etc.). As trastuzumab is expensive and associated with serious side effects (cardiotoxicity) it is important that only HER2+ patients are selected to receive it and thus it is advantageous that the methods of the current invention allow the quick and cheap detection of the HER2 status of patients.
In one embodiment, the presence of absence of the ERRB2 Exon 20 insertion mutations is detected using the methods of the current invention.
A further non-limiting list of ERBB2 mutations is shown in the table below:
EML4-ALK is an abnormal gene fusion of echinoderm microtubule-associated protein-like 4 (EML4) gene and anaplastic lymphoma kinase (ALK) gene. This gene fusion leads to the production of the protein EML4-ALK, which appears to promote and maintain the malignant behaviour of cancer cells. EML4-ALK positive lung cancer is a primary malignant lung tumour whose cells contain this mutation.
There are numerous targeted therapies approved and in development against this mutation and its associated disease(s), a non-limiting list of some such therapies can be seen in the table below:
EML4-ALK gene fusions are responsible for approximately 5% of non-small cell lung cancers (NSCLC), with about 9,000 new cases in the US per year and about 45,000 worldwide.
There are numerous variants of EML4-ALK with all variants having the essential coiled-coil domain in the EML4 N-terminal portion and in the kinase domain of ALK exon 20 that are needed for transforming activity. Fusion of exon 13 of EML4 with exon 20 of ALK (variant 1: V1) (detection of which can be seen in
V3 has emerged as a marker suitable for the selection of patients who are likely to have shorter progression-free survival (PFS) after non-tyrosine kinase inhibitor (TKI) treatment such as chemotherapy and radiotherapy. There is further evidence that V3 is associated with shorter PFS of those patients who receive first- and second-generation treatment lines and worse overall survival (OS) compared to V1 and V2 of EML4-ALK.
It has also been found that V3-positive patients develop resistance to first and second treatment lines though the development of resistance mutations and possibly facilitated by incomplete tumour cell suppression due to a higher IC50 of wild-type V3. Detection of the unfavourable V3 could be used to select patients requiring more aggressive surveillance and treatment strategies. It appears that administration of the third generation Lorlatinib to patients with V3 may confer longer PFS over those with V1 and thus it is advantageous that the methods of the current invention allow the quick and cheap detection of which variant a patient may have.
The methods of the current invention further allow the detection of resistance mutations such as, but not limited to: G1202R, G1269A, E1210K, D1203, S1206C, L1196M, F1174C, 11171T, 11171N/S, V1180L, T1151K and C1156Y.
G1202R, for example, is a solvent-front mutation which causes interference with drug binding and confers a high level of resistance to first- and second-generation ALK inhibitors. Thus it is advantageous that the methods of the current invention allow identification of those patients who may possess this mutation and benefit from treatment initiation on a third generation treatment rather than a first or second.
A further non-limiting list of EML4-ALK mutations is shown in the table below:
The identification of the epidermal growth factor receptor (EGFR) as an oncogene led to the development of targeted therapies such as gefitinib, erlotinib, afatinib, brigatinib and icotinib for lung cancer, and cetuximab for colon cancer. However, many people develop resistance to these therapies. Two primary sources of resistance are the T790M mutation and the MET oncogene.
EGFR mutations occur in EGFR exons 18-21 and mutations in exons 18, 19 and 21 and indicate suitability for treatment with EGFR-TKIs (tyrosine kinase inhibitors). Mutations in exon 20 (with the exception of a few mutations) show the tumours are EGFR-TKI resistant and not suitable for treatment with EGFR-TKIs.
The two most common EGFR mutations are short in-frame deletions of exon 19 and a point mutation (CTG to CGG) in exon 21 at nucleotide 2573, which results in substitution of leucine by arginine at codon 858 (L858R) Together, these two mutations account for ˜90% of all EGFR mutations in non-small cell lung cancer (NSCLC). Screening for these mutations in patients with NSCLC can be used to predict which patients will respond to TKIs.
Thus it is advantageous that the methods of the current invention allow identification of those patients who may possess these mutations and benefit from treatment initiation on TKIs. The person skilled in the art will appreciate that the methods of the current invention allow identification of a range of EGFR mutations, a non-exhaustive list of such mutations is: G719X, Ex19Del, S7681, Ex201 ns and L861Q.
A further non-limiting list of mutations is shown in the table below:
ROS1 is a receptor tyrosine kinase (encoded by the gene ROS1) with structural similarity to the anaplastic lymphoma kinase (ALK) protein; it is encoded by the c-ros oncogene.
A non-limiting list of ROS1 mutations is shown in the table below:
The RET proto-oncogene encodes a receptor tyrosine kinase for members of the glial cell line-derived neurotrophic factor (GDNF) family of extracellular signalling molecules.
A non-limiting list of RET mutations is shown in the table below:
MET exon 14 skipping occurs with an approximately 5% frequency in NSCLC and is seen in both squamous and adenocarcinoma histology.
A non-limiting list of MET mutations is shown in the table below:
NTRK gene fusions lead to abnormal proteins called TRK fusion proteins, which may cause cancer cells to grow. NTRK gene fusions may be found in some types of cancer, including cancers of the brain, head and neck, thyroid, soft tissue, lung, and colon. Also called neurotrophic tyrosine receptor kinase gene fusion.
A non-limiting list of NTRK mutations is shown in the table below:
In one embodiment of the invention, there is provided a panel comprising a plurality of probe molecules (A0) wherein each A0 is complementary to a target mutation. The mutation may be selected from any mutation previously, or subsequently, described or known. The person skilled in the art will thus appreciate that within the scope of the invention are included panels which may be useful in the detection of one or more mutations to the any of the proto-oncogenes or oncogenes previously, or subsequently, described or known.
In one embodiment, the panel comprises 5-500 individual probe molecules, each complementary to a specific target mutation. In one embodiment, the panel comprises 5-400 individual probe molecules, each complementary to a specific target mutation. In one embodiment, the panel comprises 5-300 individual probe molecules, each complementary to a specific target mutation. In one embodiment, the panel comprises 5-200 individual probe molecules, each complementary to a specific target mutation. In one embodiment, the panel comprises 5-100 individual probe molecules, each complementary to a specific target mutation. In one embodiment, the panel comprises 5-50 individual probe molecules, each complementary to a specific target mutation.
In one embodiment, there may be a plurality of probe molecules specific to the same mutation. In one embodiment, there may be only one probe molecule specific to each mutation of the panel.
In one embodiment, there is provided a panel, wherein the panel comprises a plurality of probe molecules wherein one or more probes are complementary to an EGFR mutation, one or more probes are complementary to a KRAS mutation, one or more probes are complementary to a ERBB2/HER2 mutation, one or more probes are complementary to a EML4-ALK mutation, one or more probes are complementary to a ROS1 mutation, one or more probes are complementary to a RET mutation and one or more probes are complementary to a MET mutation.
In one embodiment, there is provided a panel, wherein the panel comprises a plurality of probe molecules wherein one or more probes may be complementary to an EGFR mutation, one or more probes may be complementary to a KRAS mutation, one or more probes may be complementary to a ERBB2/HER2 mutation, one or more probes may be complementary to a EML4-ALK mutation, one or more probes may be complementary to a ROS1 mutation, one or more probes may be complementary to a RET mutation and one or more probes may be complementary to a MET mutation.
In one embodiment, there is provided a panel of probes selective for one or more EGFR, KRAS, BRAF, ERBB2/HER2, EML4-ALK, ROS1, RET, MET mutations.
In one embodiment, there is provided a panel of probe molecules selective for EGFR mutations.
In one embodiment, there is provided a panel of probe molecules selective for KRAS mutations.
In one embodiment, there is provided a panel of probe molecules selective for BRAF mutations.
In one embodiment, there is provided a panel of probe molecules selective for ERBB2/HER2 mutations.
In one embodiment, there is provided a panel of probe molecules selective for EML4-ALK mutations.
In one embodiment, there is provided a panel of probe molecules selective for ROS1 mutations.
In one embodiment, there is provided a panel of probe molecules selective for RET mutations.
In one embodiment, there is provided a panel of probe molecules selective for NTRK mutations.
In one embodiment, there is provided a panel of probe molecules selective for ROS1 mutations.
In one embodiment, there is provided a panel of probe molecules selective for MET exon 14 mutations.
In one embodiment, there is provided a panel comprising a plurality of probe molecules selective for one or more coding sequences (CDSs).
In one embodiment, there is provided a method of detecting one or more mutations using one or more of the previously described panels.
In one embodiment, there is provided a method of detecting the presence or absence or one or more mutations using one or more of the previously described panels.
In one embodiment, there is provided a kit comprising a panel, which may be as previously or subsequently described, in combination with one or more reagents, which may be as previously or subsequently described.
The person skilled in the art will appreciate that embodiments of kits that disclose A0, include within their scope embodiments wherein there is a panel comprising a plurality of A0.
In one embodiment, there is provided a methylation detection panel.
In one embodiment, there is provided a methylation detection kit.
Various further aspects and embodiments of the present invention will be apparent to those skilled in the art in view of the present disclosure.
There person skilled in the art will appreciate that when the term ‘infer’ is used, for example, ‘infer the presence of absence of a particular sequence or epigenetic modification’ it refers to determining the presence or absence of a particular feature based on presence of absence of A2, or copies of A2 or a region of A2 or copies of a region A2.
The person skilled in the art will further appreciate that embodiments wherein a nucleic acid analyte is selectively modified or converted, it is particular bases of the nucleic acid analyte which are selectively modified or converted. For example, the conversion of cytosine bases to uracil as a result of bisulfite treatment.
“and/or” where used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other. For example “A and/or B” is to be taken as specific disclosure of each of (i) A, (ii) B and (iii) A and B, just as if each is set out individually herein.
Unless context dictates otherwise, the descriptions and definitions of the features set out above are not limited to any particular aspect or embodiment of the invention and apply equally to all aspects and embodiments which are described.
It will further be appreciated by those skilled in the art that although the invention has been described by way of example with reference to several embodiments, it is not limited to the disclosed embodiments and that alternative embodiments could be constructed without departing from the scope of the invention as defined in the appended claims.
The person skilled in the art will understand that references to ‘partially digested strand A1’ may refer to the single-stranded oligonucleotide formed by progressive digestion of A0 when hybridised to a target analyte sequence, in the 3′-5′ direction until the strands dissociate due to lack of complementarity.
The person skilled in the art will understand that references to ‘partially double-stranded’ nucleic acids may refer to nucleic acids wherein one or more portions are double-stranded and one or more portions are single-stranded.
The person skilled in the art will understand that references to ‘substantially double-stranded’ nucleic acids may refer to nucleic acids wherein one or more portions are double-stranded and one or more smaller portions are single-stranded.
| Number | Date | Country | Kind |
|---|---|---|---|
| 1919235.0 | Dec 2019 | GB | national |
This application is the U.S. national stage filing under 35 U.S.C. § 371 of International Application No. PCT/GB2020/053363, filed on Dec. 23, 2020, which claims the benefit of priority to GB Application No. 1919235.0, filed on Dec. 23, 2019.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/GB2020/053363 | 12/23/2020 | WO |
