Patent Application
20240301481
This invention generally relates to molecular biology and high-throughput sequencing such as NexGen Sequencing (NGS) of nucleic acids from, for example, genomes. In alternative embodiments, provided are primer-based nucleic acid amplification methods capable of selecting against amplification or retrieval of a particular allele while not interfering with the amplification or retrieval of any alternative alleles or mutations at a particular nucleotide position within a target sequence, or in other words, provided are methods for selectively suppressing one allele while simultaneously amplifying any alternative allele, or, provided are methods for suppressing a wild type sequence while simultaneously amplifying a point mutation, including amplifying single nucleotide variants (SNVs), insertions and deletions. In alternative embodiments, fusions in cDNAs can also be detected. In alternative embodiments, a portion of the nucleic acid composition does not selectively suppress the amplification of a specific nucleic acid target sequence, thereby providing an internal control useful in determining the success of the amplification and in determining relative ratios of a specific nucleic target sequence to those encoding alternative allele(s) or mutations. In alternative embodiments, provided are methods for diagnosing a disease or condition comprising use of a synthetic DNA polynucleotide (referred to as a selector polynucleotide) and/or a method as provided herein. In alternative embodiments, provided are methods for treating, ameliorating or preventing a disease or condition comprising use of a synthetic DNA polynucleotide (referred to as a selector polynucleotide) and/or a method as provided herein, including diagnostic methods as provided herein.
With the advent of the Liquid Biopsy, rare allele detection has become more important than ever. Circulating cell-free DNA (ccfDNA) is present in everyone's blood, and most is derived from healthy, normal cells. However, in pregnant women, small amounts of fetal DNA are also present and can be used to look for genetic diseases in a procedure known as non-invasive prenatal testing (NIPT); similarly, in solid organ transplants (SOT), minute amounts of donor-derived cell-free DNA (dd-cfDNA) can be used to detect acute rejection for a variety of organs; and in cancer, scant levels of circulating tumor DNA (ctDNA) have been used to detect early recurrence of disease. With error correction methods (e.g., molecular indexes) that improve sensitivity, specificity, and quantitation, high-throughput NexGen Sequencing (NGS) is now an excellent detection device for all of these Liquid Biopsy applications. However, the vast majority of sequence reads are uninformative: maternal reads in NIPT; recipient reads in dd-cfDNA, and wild type reads in ccfDNA. The large number of uninformative reads diminishes throughput, increases expense, and hampers detection.
Allele-specific PCR (AS-PCR) can be used to selectively amplify the desired target (a specific allele or somatic mutation), but proof-reading polymerases cannot be used, mispriming can lead to false positives, and a different primer is needed for each desired allele. Blockers against the undesired allele can also be used, but they increase the PCR assay “footprint”, which with the short pieces of DNA in ccfDNA (median size of approximately 168 bp; shorter if derived from fetal or tumor DNA) lowers detection rate. Also, although it is desirable to dramatically reduce the number of uninformative reads, a set number of such reads is useful as an internal control for comparison and to ensure that the overall reaction worked. Neither AS-PCR or Blockers can do this reliably.
In alternative embodiments, provided are primer-based nucleic acid compositions useful in methods that selectively suppress the amplification of a specific nucleic acid target sequence while selectively amplifying alternative allele(s) or mutation(s) present in a nucleic acid (or polynucleotide, or deoxyribonucleic acid (DNA), including cDNA) sequence that differ from the suppressed target by as little as one nucleotide, allowing for analysis and measurement of the alternative allele(s) or mutation(s). In alternative embodiments, a portion of the nucleic acid composition does not selectively suppress the amplification of a specific nucleic acid target sequence, thereby providing an internal control useful in determining the success of the amplification and in determining relative ratios of a specific nucleic target sequence to those encoding alternative allele(s) or mutations.
In alternative embodiments, provided are methods for selectively suppressing the isolation of a nucleic acid target by selectively detaching a binding moiety from a nucleic acid target. Retrieval or retention of alternative alleles or mutants of the nucleic acid target which have selectively retained a binding moiety is unaffected. In alternative embodiments, a portion of the nucleic acid composition used is not selectively detached from a binding moiety, thereby providing an internal control useful in determining the success of the retrieval and in determining relative ratios of a specific nucleic target sequence to those encoding alternative allele(s) or mutations.
In alternative embodiments, allele-specific or mutation-specific or methylation-specific amplification or capture is used to determine molecular haplotypes.
In alternative embodiments, the difference is detected by sequencing, hybridization, mass spectrometry, or any other technology that can detect sequence changes, including those with nucleotide resolution.
In alternative embodiments, provided are synthetic DNA polynucleotides (referred to as a selector polynucleotide) comprising at least a single residue (referred to as a selector nucleotide) that is located at the first (ultimate), second (penultimate), third (antepenultimate), fourth (preantepenultimate), fifth (propreantepenultimate), or sixth (one before the propreantepenultimate) position in reference to a 3′ end, or is at a position that is even more distal to the 3′ end,
In alternative embodiments of synthetic DNA polynucleotides (or selector polynucleotides) as provided herein:
In alternative embodiments, provided are nucleic acid amplification methods for differentiating a first nucleic acid sequence from a second nucleic acid sequence wherein the first and the second nucleic acid are in the same amplification reaction mixture, comprising:
In alternative embodiments, provided are nucleic acid amplification methods for differentiating a first nucleic acid sequence from a second nucleic acid sequence wherein the first and the second nucleic acid are in the same amplification reaction mixture, comprising:
In alternative embodiments, methods as provided herein:
In alternative embodiments, provided are kits or products of manufacture comprising materials, optionally enzymes and/or synthetic DNA polynucleotides, optionally selector polynucleotides (optionally one or a plurality of synthetic DNA polynucleotides (or selector polynucleotides) as provided herein), for practicing a method as provided or described herein, and optionally further comprising instructions for practicing a method as provided or described herein.
In alternative embodiments, provided are kits comprising materials for practicing methods as provided herein, and optionally also comprising instructions for practicing methods as provided herein.
In alternative embodiments, provided are methods for diagnosing a disease or a condition comprising determining if an individual in need thereof has the disease or condition by determining the presence or absence of an allele or a genomic sequence associated with or diagnostic of the disease or condition, wherein the presence or absence of the allele or genomic sequence associated with or diagnostic of the disease or condition is determined by using a method as provided herein. The disease can be a cancer.
In alternative embodiments, provided are methods for treating, ameliorating or preventing a disease or a condition comprising treating an individual in need thereof with a drug, drug combination or treatment regimen indicated for the disease or condition, wherein the individual in need thereof is diagnosed as having, or predisposed to having, the disease or condition using a diagnostic method as provided herein. The disease can be a cancer, or the condition is an inherited disease or genetic condition.
In alternative embodiments, provided are uses of a synthetic DNA polynucleotide as provided herein for diagnosing a disease or a condition, wherein the disease or condition is diagnosed by the presence of an allele or genomic sequence associated with or diagnostic of the disease or condition, and the presence or absence of the allele or genomic sequence associated with or diagnostic of the disease or condition is determined by using a method as provided herein.
In alternative embodiments, synthetic DNA polynucleotides as provided herein are for use in diagnosing a disease or a condition, wherein the disease or condition is diagnosed by the presence of an allele or genomic sequence associated with or diagnostic of the disease or condition, and the presence or absence of the allele or genomic sequence associated with or diagnostic of the disease or condition is determined by using a method as provided herein.
In alternative embodiments, provided are methods and synthetic DNA polynucleotides for detecting the presence or absence of a rare allele in a biological specimen, comprising using a method as provided herein, wherein optionally the biological specimen comprises or is derived from a biopsy or tissue or blood sample, or liquid sample, from an individual in need thereof.
In alternative embodiments, detecting the presence or absence of the rare allele in the biological specimen is for non-invasive pre-natal testing (NIPT), or to assess tissue compatibility or detecting donor-derived nucleic acid following organ transplant (optionally solid organ or bone marrow transplant), or to assess anti-microbial resistance (AMR) or early detection of microbial resistance in the individual in need thereof, or assessing the presence of minimum residual disease (MRE), optionally assessing MRE after bone marrow ablation.
The details of one or more exemplary embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.
All publications, patents, patent applications cited herein are hereby expressly incorporated by reference in their entireties for all purposes.
The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.
The drawings set forth herein are illustrative of exemplary embodiments provided herein and are not meant to limit the scope of the invention as encompassed by the claims.
Like reference symbols in the various drawings indicate like elements.
In alternative embodiments, provided are nucleic acid amplification methods which can select against a particular allele while not interfering with the amplification or retrieval of any alternative alleles or mutations at a particular nucleotide position within a target sequence, for example, a target sequence in a genome. In alternative embodiments, provided are nucleic acid amplification methods for suppressing a wild type sequence while simultaneously amplifying a point mutation, including single nucleotide variants, insertions, and deletions; and in the case of cDNA also including fusions.
In alternative embodiments, provided are methods that are “tunable”, meaning that an unwanted allele can be suppressed (in an amplification or retrieval process) by a factor of 10 or 100 or by whatever amount desired. This reduces the number of uninformative reads, which increases throughput, lowers the cost per sample, and since there is reduced competition for sequencing space, enhances detection of desired targets.
In alternative embodiments, methods as provided herein have the ability to allow some uninformative reads, which is unique and allows for an internal control to demonstrate that the assay worked (as opposed to a “no result” when alternative alleles or mutants are not present in the sample) and can also function as an internal standard for quantitation.
In alternative embodiments, methods as provided herein can work with any primer-based approach, and by using a method as provided herein no variant sequences are introduced into the amplification reaction. By way of example, if one were looking for a somatic mutation, the wild type allele might be a G, and possible mutant alleles would be A, C, and T; by using an amplification method as provided herein the G-containing reads would be reduced (e.g., 100-fold) without diminishing mutant reads and any of the possible mutants would be amplified by the same single primer. The number of wild type reads is markedly reduced making mutant reads more likely to be sequenced and detected, if present, and if they are absent, the set number of wild type reads assures one that the reaction worked.
In alternative embodiments, methods as provided herein can have distinct advantages over current methods, including never introducing primers that contain the sequence that one is interested in detecting; a mutant is only detected if:
In alternative embodiments, methods as provided herein can have the advantage where a single primer detects any variant at a particular nucleotide position. In contrast, allele-specific PCR needs a specific primer for each allele variant or mutation; for example, see van Mansfeld A D, Bos J L. PCR-based approaches for detection of mutated ras genes. PCR Methods Appl. 1992 May; 1(4):211-6; Darawi, M. N, Ai-Vym, C, Ramasamy, K. et al. Allele-specific polymerase chain reaction for the detection of Alzheimer's disease-related single nucleotide polymorphisms. BMC Med Genet 14, 27 (2013); Lang A H, Drexel H. Geller-Rhomberg S, et al. Optimized allele-specific real-time PCR assays for the detection of common mutations in KRAS and BRAF. J Mol Diagn. 2011; 13(1):23-28; Dobosy J R, Rose S D, et al. (2011) RNase H-dependent PCR (rhPCR): improved specificity and single nucleotide polymorphism detection using blocked cleavable primers BMC Biotechnol, 11.80.
In alternative embodiments, provided are methods comprising use of DNA amplification techniques such as polymerase chain reaction (PCR) or other primer-based amplification method.
Any known protocol or materials (including armamentarium or enzymes) used to practice DNA amplification techniques can be used to practice methods as provided herein, including for example using a proofreading enzyme with a 5′ to 3′ extension activity and having a 3′ to 5′ exonuclease activity, and/or a DNA polymerase enzyme having 5′ to 3′ extension activity and an enzyme having 3′ to 5′ exonuclease activity. Standard materials and methods for polymerase chain reactions as used to practice methods as provided herein can be found for example: in Dieffenbach and Dveksler (1995) PCR Primer: A Laboratory Manual, Cold Spring Harbor Laboratory Press, and in McPherson at al. (2000) PCR—Basics: From Background to Bench, First Edition, Springer Verlag, Germany.
An exemplary PCR protocol is shown in the following two tables, the first shows the reaction set up, the second the cycling conditions:
A variety of DNA concentrations may be used, as one practiced in the art would know. Similarly, primer concentrations can vary. Any source of Pyrococcus abyssi RNase H2 and/or other thermostable RNase H2 enzymes can be used. Thermostable proofreading DNA polymerases from other sources may be used, including, but not limited to, Q5® HOT START HIGH-FIDELITY® DNA Polymerase from New England Biolabs, and PLATINUM SUPERFI II® DNA Polymerase-High-Fidelity PCR Enzyme from ThermoFisher.
The following are general exemplary conditions for the cycling conditions and are not intended to limit the practice of amplification: denaturation temperatures in the range of 94° C. to 98° C. with times ranging from 10 seconds to 30 seconds; annealing temperatures in the range of 55° C. to 72° C. for 5 seconds to 30 seconds; extension temperatures in the range of 55° C. to 72° C. or higher for 15 seconds to 30 seconds per kilobase of DNA to be amplified. A general discussion of PCR methodologies that can be used to practice methods as provided herein can be found for example on Wikipedia (https://en.wikipedia.org/wiki/Polymerase_chain_reaction, or Valones M A, Guimarães R L, Brandão L A, de Souza P R, de Albuquerque Tavares Carvalho A, Crovela S. Principles and applications of polymerase chain reaction in medical diagnostic fields: a review. Braz J Microbiol. 2009; 40(1): 1-11; Garibyan L, Avashia N. Polymerase chain reaction. J Invest Dermatol. 2013; 133(3): 1-4; Dieffenbach C W, Lowe T M, Dveksler G S. General concepts for PCR primer design. PCR Methods Appl. 1993 Dec. 3(3):S30-7). Exemplary conditions for use of RNase H2 enzyme on the IDT web site (https://www.idtdna.com/pages/products/qpcr-and-pcr/master-mixes-reagents/rnase-h-enzyme), and also are included in Dobosy J R, Rose S D, et al. (2011) RNase H-dependent PCR (rhPCR): improved specificity and single nucleotide polymorphism detection using blocked cleavable primers, BMC Biotechnol, 11:80. Exemplary enzymes that can be used to practice methods as provided herein include for example Phusion Hot Start II®, a commercially available thermostable DNA polymerase, one of many that are available and can be used to practice methods as provided herein, see for example, Ishino S, Ishino Y, DNA polymerases as useful reagents for biotechnology—the history of developmental research in the field, Front Microbiol. 2014 Aug. 29; 5:465). Exemplary conditions for Phusion Hot Start II® are available through the ThermoFisher web site (https://www.thermofisher.com/order/catalog/product/F549L #/F549L).
In alternative embodiments, provided are methods comprising use of digital PCR, see for example Morley A A. Digital PCR: A brief history. Biomol Detect Quantif. 2014 Aug. 15; 1(1): 1-2, or DigitalPCR, or dPCR, including for example, Droplet Digital PCR (ddPCR), for use in methods as provided herein, for example, for the production of amplicons of interest, or for producing new extended DNA polynucleotides; and methods as provided herein can be practiced using any method known in the art, for example as described by U.S. Pat. No. 9,797,007.
In alternative embodiments, provided are synthetic DNA polynucleotides comprising at least a single ribonucleotide residue (referred to as a selector nucleotide) that can be located at the first (ultimate), second (penultimate), third (antepenultimate), fourth (preantepenultimate), fifth (propreantepenultimate), or sixth (one before the propreantepenultimate) position in reference to a 3′ end of the synthetic DNA polynucleotide (or selector nucleotide), and in alternative embodiments, a second or third or additional ribonucleotide residues are included in the provided in the synthetic DNA polynucleotide.
In alternative embodiments, selector polynucleotides are used as primers in amplification methods capable of selecting against amplification of a particular allele while not interfering with the amplification or retrieval of any alternative alleles or mutations at a particular nucleotide position within a target sequence. The design, composition, and manufacture of the selector polynucleotide is based on the allele that is to be suppressed. By way of example, if one is interested in single nucleotide mutations occurring at c.38 in the human oncogene KRAS, a chimeric DNA/RNA polynucleotide that is a perfect match to the wild type sequence and that contains a single ribonucleotide at the position corresponding to the site of interest would be created. Referring to
As described in Examples, the design, composition, and manufacture of selector polynucleotides for the suppression of wild type sequences without interfering with the detection of other alternatives, including, but not limited to, deletions, insertions, and in the case of cDNA, fusions, may also be accomplished. Since suppression is based on the sequence of the allele to be suppressed, alternative designs can be accomplished by one skilled in the art.
In alternative embodiments, provided are methods wherein the sequence of an amplicon, or a new extended DNA polynucleotide, is determined by DNA sequencing, optionally using a method comprising use of Sanger sequencing, or next generation sequencing (NGS), single molecule real time (SMRT) sequencing, nanopore DNA sequencing, reversible terminated chemistry (for example, SOLEXA technology (Illumina)), combinatorial probe anchor synthesis (cPAS), mass spectrometry sequencing, or massively parallel signature sequencing (MPSS) or any equivalent thereof or any combination thereof.
Any sequencing method known in the art can be used to sequence an amplicon, or a new extended DNA polynucleotide, produced by a method as provided herein.
Provided are products of manufacture and kits for practicing methods as provided herein; and optionally, products of manufacture and kits can further comprise instructions for practicing methods as provided herein.
Any of the above aspects and embodiments can be combined with any other aspect or embodiment as disclosed here in the Summary, Figures and/or Detailed Description sections.
As used in this specification and the claims, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise.
Unless specifically stated or obvious from context, as used herein, the term “or” is understood to be inclusive and covers both “or” and “and”.
Unless specifically stated or obvious from context, as used herein, the term “about” is understood as within a range of normal tolerance in the art, for example within 2 standard deviations of the mean. About can be understood as within 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear from the context, all numerical values provided herein are modified by the term “about.”
Unless specifically stated or obvious from context, as used herein, the terms “substantially all”, “substantially most of”, “substantially all of” or “majority of” encompass at least about 90%, 95%, 97%, 98%, 99% or 99.5%, or more of a referenced amount of a composition.
The entirety of each patent, patent application, publication and document referenced herein hereby is incorporated by reference. Citation of the above patents, patent applications, publications and documents is not an admission that any of the foregoing is pertinent prior art, nor does it constitute any admission as to the contents or date of these publications or documents. Incorporation by reference of these documents, standing alone, should not be construed as an assertion or admission that any portion of the contents of any document is considered to be essential material for satisfying any national or regional statutory disclosure requirement for patent applications. Notwithstanding, the right is reserved for relying upon any of such documents, where appropriate, for providing material deemed essential to the claimed subject matter by an examining authority or court.
Modifications may be made to the foregoing without departing from the basic aspects of the invention. Although the invention has been described in substantial detail with reference to one or more specific embodiments, those of ordinary skill in the art will recognize that changes may be made to the embodiments specifically disclosed in this application, and yet these modifications and improvements are within the scope and spirit of the invention. The invention illustratively described herein suitably may be practiced in the absence of any element(s) not specifically disclosed herein. Thus, for example, in each instance herein any of the terms “comprising”, “consisting essentially of”, and “consisting of” may be replaced with either of the other two terms. Thus, the terms and expressions which have been employed are used as terms of description and not of limitation, equivalents of the features shown and described, or portions thereof, are not excluded, and it is recognized that various modifications are possible within the scope of the invention. Embodiments of the invention are set forth in the following claims.
The invention will be further described with reference to the examples described herein; however, it is to be understood that the invention is not limited to such examples.
Unless stated otherwise in the Examples, all recombinant DNA techniques are carried out according to standard protocols, for example, as described in Sambrook et al. (2012) Molecular Cloning: A Laboratory Manual, 4th Edition, Cold Spring Harbor Laboratory Press, NY and in Volumes 1 and 2 of Ausubel et al. (1994) Current Protocols in Molecular Biology, Current Protocols, USA. Other references for standard molecular biology techniques include Sambrook and Russell (2001) Molecular Cloning: A Laboratory Manual, Third Edition, Cold Spring Harbor Laboratory Press, NY, Volumes I and II of Brown (1998) Molecular Biology LabFax, Second Edition, Academic Press (UK). Standard materials and methods for polymerase chain reactions can be found in Dieffenbach and Dveksler (1995) PCR Primer: A Laboratory Manual, Cold Spring Harbor Laboratory Press, and in McPherson at al. (2000) PCR-Basics: From Background to Bench, First Edition, Springer Verlag, Germany.
In alternative embodiments, the differences noted in the Examples is detected by sequencing, hybridization, mass spectrometry, or any other technology that can detect sequence changes, including those with nucleotide resolution.
This example describes and demonstrates the efficacy of exemplary methods as provided herein for selectively suppressing one allele while simultaneously amplifying any alternative allele. This example also describes and demonstrates the efficacy of exemplary methods for suppressing a wild type sequence while simultaneously amplifying a point mutation.
In alternative embodiments, exemplary methods as provided herein comprise suppressing the exponential amplification of KRAS DNA that codes for the wild type “G” nucleotide at coding position 35 (in this example, KRAS c.35) while simultaneously allowing exponential amplification of any of the three mutations possible at this position. Mutations at this position are the most frequent cancer-causing KRAS mutations representing approximately 65% of the point mutations in KRAS codons 12 and 13. The selector primer used would contain a “G” ribonucleotide (riboG or rG) at the antepenultimate position, be a perfect match to the wild type template, and be extended by the proofreading DNA polymerase enzyme. Since the newly formed duplex DNA retains the riboG, thermostable ribonuclease H2 (TS RNase H2), present in the reaction, cuts at the 5′ side of the ribonucleotide and detaches most of the primer sequence; exponential amplification is suppressed. In contrast, templates that code for the c.35G>A (p.G12D) mutation; or c.35G>T (p.G12V) mutation; or c.35G>C (p.G12A) mutation are not a match for the selector primer at the antepenultimate position, resulting in the 3′ to 5′ exonuclease activity of the proofreading polymerase digesting the primer back through the mismatch and the ribonucleotide is eliminated prior to extension of the primer over the mutant site. The loss of the ribonucleotide makes the incorporated primer sequence resistant to removal by ribonuclease. The return primer copies over the incorporated (i.e., partially digested, then extended) first primer and any mutant sequence is exponentially amplified.
Non-limiting examples of a proofreading DNA polymerase that can be used in methods as provided herein include Phusion Hot Start II DNA Polymerase (ThermoFisher Scientific, cat. #F549L); Q5® Hot Start High-Fidelity DNA Polymerase (New England Biolabs, cat. #M0493L); and Platinum SuperFi II DNA Polymerase (ThermoFisher Scientific, cat. #12361050). A non-limiting example of a thermostable ribonuclease H2 that can be used in methods as provided herein is RNase H2 enzyme (IDT, cat. #Nov. 3, 2002-03).
This exemplary method includes distinct advantages over current methods, including never introducing primers that contain the sequence that one is interested in detecting; a mutant is only detected if: 1. The mismatch between selector primer and mutated template is recognized and removed by the 3′ to 5′ exonuclease activity of the proofreading polymerase, and 2. If the high-fidelity polymerase copies over the mutated template. In contrast, allele-specific amplification introduces the variant sequence that one is interested in detecting, which can lead to false positives. Another advantage is that a single primer detects any variant at a particular nucleotide position; in contrast, allele-specific PCR needs a specific primer for each allele variant or mutation.
This example demonstrates exemplary methods for suppressing the amplification of a wild type DNA while simultaneously amplifying a template that contains a deletion.
In alternative embodiments, exemplary methods as provided herein comprise suppressing the exponential amplification of wild type epidermal growth factor receptor (EGFR) DNA while simultaneously allowing exponential amplification of DNA with the inframe deletion, c.2235_2249 del (p.E746_A750del). Inframe deletions represent approximately 41% of all EGFR mutations in cancer, and this particular mutation is among the more frequent ones.
The 3′ end of the selector primer ends with GAA, contains a “G” ribonucleotide (riboG or rG) at the antepenultimate position, and is a perfect match for the wild type EGFR codon that codes for the glutamic acid residue at amino acid position 746. Since the newly formed duplex DNA retains the riboG, thermostable ribonuclease H2 (TS RNase H2), present in the reaction, cuts at the 5′ side of the ribonucleotide and detaches most of the primer sequence, resulting in having exponential amplification suppressed.
In contrast, templates for the c.2235_2249 del (p.E746_A750del) deletion are not a match for the selector primer at the antepenultimate position, resulting in the 3′ to 5′ exonuclease activity of the proofreading polymerase digesting the primer back through the mismatch and the ribonucleotide is eliminated prior to extension of the primer over the mutant site. The loss of the ribonucleotide makes the incorporated primer sequence resistant to removal by ribonuclease. The return primer copies over the incorporated (i.e., partially digested, then extended) first primer and any mutant sequence is exponentially amplified.
Non-limiting examples of a proofreading polymerase that can be used to practice methods as provided herein includes Phusion Hot Start II DNA Polymerase (ThermoFisher Scientific, cat. #F549L); Q5® Hot Start High-Fidelity DNA Polymerase (New England Biolabs, cat. #M0493L); and Platinum SuperFi II DNA Polymerase (ThermoFisher Scientific, cat. #12361050). A non-limiting example of a thermostable ribonuclease H2 that can be used to practice methods as provided herein is RNase H2 enzyme (IDT, cat. #Nov. 3, 2002-03).
This exemplary method includes distinct advantages over current methods, including never introducing primers that contain the sequence that one is interested in detecting; in contrast, allele-specific amplification introduces the variant sequence that one is interested in detecting, which can lead to false positives. Another advantage is that a single primer detects any variant at a particular nucleotide position; in contrast, allele-specific PCR needs a specific primer for each allele variant or mutation.
This example demonstrates exemplary methods for suppressing the amplification of a wild type DNA while simultaneously amplifying a template that contains an insertion.
A non-limiting example would be suppressing the exponential amplification of wild type EGFR DNA while simultaneously allowing exponential amplification of DNA with the c.2300_2308 dup (p.A767_V769dup) insertion. This insertion is a duplication, and patients with it often have decreased sensitivity to first- and second-generation EGFR tyrosine kinase inhibitors (TKIs). The 3′ end of the selector primer ends with ACA, contains an “A” ribonucleotide (riboA or rA) at the antepenultimate position, and is a perfect match for the wild type EGFR codon that codes for the aspartic acid residue at amino acid position 770. Since the newly formed duplex DNA retains the riboA, thermostable ribonuclease H2 (TS RNase H2), present in the reaction, cuts at the 5′ side of the ribonucleotide and detaches most of the primer sequence; exponential amplification is suppressed. In contrast, templates containing the c.2300_2308 dup (p.A767_V769dup) insertion are not a match for the selector primer at the antepenultimate position, resulting in the 3′ to 5′ exonuclease activity of the proofreading polymerase digesting the primer back through the mismatch and the ribonucleotide is eliminated prior to extension of the primer over the mutant site. The loss of the ribonucleotide makes the incorporated primer sequence resistant to removal by ribonuclease. The return primer copies over the incorporated (i.e., partially digested, then extended) first primer and any mutant sequence is exponentially amplified.
Non-limiting examples of a proofreading polymerase that can be used to practice methods as provided herein includes Phusion Hot Start II DNA Polymerase (ThermoFisher Scientific, cat. #F549L); Q5® Hot Start High-Fidelity DNA Polymerase (New England Biolabs, cat. #M0493L); and Platinum SuperFi II DNA Polymerase (ThermoFisher Scientific, cat. #12361050). A non-limiting example of a thermostable ribonuclease H2 that can be used to practice methods as provided herein is RNase H2 enzyme (IDT, cat. #Nov. 3, 2002-03).
This exemplary method includes distinct advantages over current methods, including never introducing primers that contain the sequence that one is interested in detecting; in contrast, allele-specific amplification introduces the variant sequence that one is interested in detecting, which can lead to false positives. Another advantage is that a single primer detects any variant at a particular nucleotide position; in contrast, allele-specific PCR needs a specific primer for each allele variant or mutation.
This example demonstrates exemplary methods for suppressing the amplification of a wild type cDNA while simultaneously amplifying a template that codes for a fusion protein.
A non-limiting example would be suppressing the exponential amplification of wild type echinoderm microtubule-associated protein-like 4 (EML4) while simultaneously allowing exponential amplification of a cDNA that codes for an EML4-ALK fusion protein (ALK stands for Anaplastic lymphoma kinase, also known as ALK tyrosine kinase receptor or CD246). EML4-ALK fusion protein has been found in non-small cell lung cancer (NSCLC) and is the target for Crizotinib, among other targeted therapies. A cDNA preparation is first made, followed by amplification using a selector primer with the sequence GTG at its 3′ end, where the antepenultimate “G” is a ribonucleotide (riboG or rG) and is a perfect match for the wild type EML4 sequence. Since the newly formed duplex DNA retains the riboG, thermostable ribonuclease H2 (TS RNase H2), present in the reaction, cuts at the 5′ side of the ribonucleotide and detaches most of the primer sequence; exponential amplification is suppressed. In contrast, EML4-ALK fusion templates are not a match for the selector primer at the antepenultimate position, resulting in the 3′ to 5′ exonuclease activity of the proofreading polymerase digesting the primer back through the mismatch and the ribonucleotide is eliminated prior to extension of the primer over the mutant site. The loss of the ribonucleotide makes the incorporated primer sequence resistant to removal by ribonuclease. The return primer copies over the incorporated (i.e., partially digested, then extended) first primer and any mutant sequence is exponentially amplified. Non-limiting examples of a proofreading polymerase include Phusion Hot Start II DNA Polymerase (ThermoFisher Scientific, cat. #F549L); Q5® Hot Start High-Fidelity DNA Polymerase (New England Biolabs, cat. #M0493L); and Platinum SuperFi II DNA Polymerase (ThermoFisher Scientific, cat. #12361050).
A non-limiting example of a thermostable ribonuclease H2 that can be used to practice methods as provided herein is RNase H2 enzyme (IDT, cat. #Nov. 3, 2002-03). A non-limiting example of a reverse transcriptase used to make the cDNA from an mRNA template that can be used to practice methods as provided herein is SuperScript™ III Reverse Transcriptase (ThermoFisher Scientific, cat. #18080085).
This exemplary method includes distinct advantages over current methods, including never introducing primers that contain the sequence that one is interested in detecting; in contrast, allele-specific amplification introduces the variant sequence that one is interested in detecting, which can lead to false positives.
This example demonstrates exemplary methods for suppressing the amplification of one allele while simultaneously amplifying any alternative allele present at the nucleotide position, specifically, when one or the other allele has been created based on the presence or absence of a nucleotide modification. A non-limiting example would be when an unmodified cytosine nucleotide is changed into a deoxyuridine (dU) while a methylated or hydroxymethylated cytosine is not.
A non-limiting example would be use of a selector primer that contains a “G” ribonucleotide (riboG or rG) at the antepenultimate position, is a perfect match to the treated template (which in the complementary position remains a “C” since it is modified and therefore not changed), and is extended by a proofreading enzyme. Since the newly formed duplex DNA retains the riboG, thermostable ribonuclease H2 (TS RNase H2), present in the reaction, cuts at the 5′ side of the ribonucleotide and detaches most of the primer sequence; exponential amplification is suppressed. In contrast, in treated templates where the “C” has been changed to “dU” there is not a match for the selector primer at the antepenultimate position, resulting in the 3′ to 5′ exonuclease activity of the proofreading polymerase digesting the primer back through the mismatch and the ribonucleotide is eliminated prior to extension of the primer over the “dU” site (pairing an “A” with it). The loss of the ribonucleotide makes the incorporated primer sequence resistant to removal by ribonuclease. The return primer copies over the incorporated (i.e., partially digested, then extended) first primer and the “dU” sequence is exponentially amplified (being changed to a “T” during amplification).
A non-limiting example would be the converse of the aforementioned, where the selector primer now contains an “A” ribonucleotide (riboA or rA) at the antepenultimate position, is a perfect match to the treated template (which in the complementary position becomes a “dU” since it was unmodified and therefore changed), and is extended by a proofreading enzyme. Since the newly formed duplex DNA retains the riboA, thermostable ribonuclease H2 (TS RNase H2), present in the reaction, cuts at the 5′ side of the ribonucleotide and detaches most of the primer sequence; exponential amplification is suppressed. In contrast, in treated templates where the “C” has not been changed (protected by the modification) there is not a match for the selector primer at the antepenultimate position, resulting in the 3′ to 5′ exonuclease activity of the proofreading polymerase digesting the primer back through the mismatch and the ribonucleotide is eliminated prior to extension of the primer over the mutant site. The loss of the ribonucleotide makes the incorporated primer sequence resistant to removal by ribonuclease. The return primer copies over the incorporated (i.e., partially digested, then extended) first primer and the sequence is exponentially amplified.
Non-limiting examples of a proofreading polymerase that can be used to practice methods as provided herein include Phusion Hot Start II DNA Polymerase (ThermoFisher Scientific, cat. #F549L); Q5® Hot Start High-Fidelity DNA Polymerase (New England Biolabs, cat. #M0493L); and Platinum SuperFi II DNA Polymerase (ThermoFisher Scientific, cat. #12361050).
This exemplary method includes distinct advantages over current methods, including never introducing primers that contain the sequence that one is interested in detecting; in contrast, allele-specific amplification introduces the variant sequence that one is interested in detecting, which can lead to false positives.
This example demonstrates exemplary methods for suppressing the amplification of one allele while simultaneously amplifying any alternative allele present at the nucleotide position along with alleles located on the same DNA homologue as the alternative allele thus allowing molecular haplotyping.
A non-limiting example would be determining the presence or absence of the AGT haplotype in the ITGA4 gene. Single nucleotide polymorphisms within the ITGA4 gene include c.1845G>A, c.2633A>G, and c.2883C>T and the haplotype AGT is associated with the development of antibody-mediated rejection in heart transplant patients. If genotyping reveals an individual is heterozygous at two or three of the sites, then haplotypes can be determined using a selector primer to an appropriate heterozygous site and a return primer located beyond the most distal heterozygous site. By way of example, if a DNA is heterozygous at c. 1845, then the selector primer would contain a “G” ribonucleotide (riboG or rG) at the antepenultimate position, corresponding to the c. 1845 position; the return primer would be downstream of the c.2883 position. This would lead to the suppression of the c. 1845G allele while allowing exponential amplification of the c. 1845A allele. Because of the high fidelity and the very low level of strand-switching of the proofreading enzyme used, alleles present on the same DNA homologue that is exponentially amplified can be determined and the haplotype found for the 1845A DNA homologue. The haplotype for the other homologue can be inferred from the genotype or confirmed in a separate reaction using a selector primer containing an “A” ribonucleotide (riboA or rA) at the antepenultimate position.
Non-limiting examples of a proofreading polymerase that can be used to practice methods as provided herein includes Phusion Hot Start II DNA Polymerase (ThermoFisher Scientific, cat. #F549L); Q5® Hot Start High-Fidelity DNA Polymerase (New England Biolabs, cat. #M0493L); and Platinum SuperFi II DNA Polymerase (ThermoFisher Scientific, cat. #12361050). A non-limiting example of a thermostable ribonuclease H2 that can be used to practice methods as provided herein is RNase H2 enzyme (IDT, cat. #Nov. 3, 2002-03).
This exemplary method includes distinct advantages over current methods, including never introducing primers that contain the sequence that one is interested in detecting; in contrast, allele-specific amplification introduces the variant sequence that one is interested in detecting, which can lead to false positives. Another advantage is that a single primer detects any variant at a particular nucleotide position; in contrast, allele-specific PCR needs a specific primer for each allele variant or mutation.
This example demonstrates exemplary methods for partially suppressing the amplification of one allele while simultaneously amplifying any alternative allele present at the nucleotide position. This can also be applied to the partial suppression of a wild type sequence while simultaneously amplifying a point mutation. Referring to the latter case, by only partially suppressing the amplification of the wild type while not interfering with the amplification of any mutant, two outcomes can be accomplished: 1. If no mutant is present, then the partial amplification of the wild type provides an internal control that confirms that the overall reaction worked; and 2. By controlling the level of suppression of the wild type an internal standard is provided to which the level of amplification of the mutant (if present) can be compared.
A non-limiting example would be partially suppressing the exponential amplification of KRAS DNA that codes for the wild type “G” nucleotide at coding position 35 while simultaneously allowing exponential amplification of any of the three mutations possible at this position. The selector primer would contain a “G” ribonucleotide (riboG or rG) at the antepenultimate position, be a perfect match to the wild type template, and be extended by the proofreading enzyme. Mixed in with the selector primer at a predetermined amount would be a counterpart that is identical to the selector primer except the “G” at the antepenultimate position would not be a ribonucleotide, but a normal deoxyribonucleotide. Newly formed duplex DNA that retains the riboG would be cut by thermostable ribonuclease H2 (TS RNase H2) present in the reaction, which detaches most of the primer sequence; exponential amplification is suppressed. However, newly formed duplex DNA that contains the counterpart to the selector primer (i.e., primer containing the normal deoxynucleotide counterpart at the selector nucleotide position) would not be cut and the entirety of the primer retained. Exponential amplification of the wild type would be only partially suppressed. Templates that code for the c.35G>A (p.G12D) mutation; or c.35G>T (p.G12V) mutation; or c.35G>C (p.G12A) mutation are not a match for the selector primer (or its counterpart) at the antepenultimate position, resulting in the 3′ to 5′ exonuclease activity of the proofreading polymerase digesting the primer back through the mismatch and the ribonucleotide or its deoxyribonucleotide counterpart is eliminated prior to extension of the primer over the mutant site. The absence of any ribonucleotide makes the incorporated primer sequence resistant to removal by ribonuclease. The return primer copies over the incorporated (i.e., partially digested, then extended) first primer and any mutant sequence is exponentially amplified.
Non-limiting examples of a proofreading polymerase that can be used to practice methods as provided herein include Phusion Hot Start II DNA Polymerase (ThermoFisher Scientific, cat. #F549L); Q5® Hot Start High-Fidelity DNA Polymerase (New England Biolabs, cat. #M0493L); and Platinum SuperFi II DNA Polymerase (ThermoFisher Scientific, cat. #12361050). A non-limiting example of a thermostable ribonuclease H2 that can be used to practice methods as provided herein is RNase H2 enzyme (IDT, cat. #Nov. 3, 2002-03).
This exemplary method includes distinct advantages over current methods, including never introducing primers that contain the sequence that one is interested in detecting; a mutant is only detected if: 1. The mismatch between selector primer and mutated template is recognized and removed by the 3′ to 5′ exonuclease activity of the proofreading polymerase, and 2. If the high-fidelity polymerase copies over the mutated template. In contrast, allele-specific amplification introduces the variant sequence that one is interested in detecting, which can lead to false positives. Another advantage is that a single primer detects any variant at a particular nucleotide position; in contrast, allele-specific PCR needs a specific primer for each allele variant or mutation. Another advantage as described in this Example and applicable to the other Examples, and generally to all methods as provided herein, is the ability to allow a set amount of amplification of a particular allele or wild type that serves as an internal control demonstrating that the reaction has worked; and as an internal standard to which the level of amplification of the alternative allele or mutant can be compared (if present).
A number of embodiments of the invention have been described. Nevertheless, it can be understood that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, other embodiments are within the scope of the following claims.
This patent Convention Treaty (PCT) International Application claims the benefit of priority under 35 U.S.C. § 119(e) of U.S. Provisional Serial Application No. (USSN) 63/144,723, filed Feb. 2, 2021. The aforementioned application is expressly incorporated herein by reference in its entirety and for all purposes. All publications, patents, patent applications cited herein are hereby expressly incorporated by reference for all purposes.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2022/014908 | 2/2/2022 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63144723 | Feb 2021 | US |
