Patent Application
20180187246
This application relates to the field of molecular pathology (for example, cancer diagnosis, prognosis, treatment and/or therapy prediction) through the detection of RNA, mutations, copy number changes and determination of the methylation status of specific sequences of the genome of individual patients in hybridization assays (southern blot, ISH, dot blot) including in situ determination of the methylation status of specific sequences of the genome of individual patients in individual cells. More specifically, this disclosure relates to: a) target-specific probes covalently-attached to a labeled tail, b) the synthesis method of the probe, c) the usage of the probe such as an in situ hybridization-based method to correlate the methylation status of a promoter region of a gene in a biopsy or cytology specimen of a patient to the morphology and localization in that specimen, and d) kits comprising the target-specific probes. The latter method and products allow detection of (epi) genetic changes in specific cell types of histological or cytological (cancer) specimens or on membranes that will contribute to scientific research and that will help physicians to accurately diagnose diseases and/or start an appropriate treatment.
The following target-specific probes or signal amplification systems for in situ hybridization have been described:
This method combines PCR amplification for sufficient signal amplification and enzymatic restriction to allow probe access; and does thus not allow quantification of the target at physiological levels.
In RCA, the target DNA is restriction digested at the 3′ end of the target sequence and irreversibly made single-stranded by strand-specific 5′-3′ exonucleolysis. Padlock probes are hybridized to their target sequences and the probe ends are joined through ligation, locking the probe onto the target molecule. After ligation, the RCA is initiated by the F29 DNA polymerase by turning the target molecule into a primer through 3′-5′ exonucleolysis of any 3′ end protruding beyond the padlock probe hybridization site. The padlock probe then serves as the template for DNA synthesis. The RCA product is detected through hybridization of fluorescence-labeled oligonucleotides to tag sequences, specific for the padlock probe (Larsson et al., 2004).
The sensitivity of this technique is only 10% since the enzymatic restriction step that exposes the sense or anti-sense strand is not absolute and is difficult to regulate; further factors contributing to the reduced sensitivity is the low PCR efficiency and DNA loss.
RCA is thus based on amplification of the target-specific probe and detection of the amplified material.
Moreover, the detection probes used in RCA are molecular inversion probes (MIP).
The probes are designed with complementary sequences to the target at its 5′ and 3′ ends. The internal region contains two universal PCR primer sites that are common to all MIPs, as well as a probe-release site, which is usually a restriction site.
A lollipop oligomer is a branched oligomer that comprises a tail portion, a right arm portion, and a left arm portion. The two arms each end with sequences complementary to adjacent sequences in a target sequence. This allows the right and left arms to be ligated together when the oligomer is hybridized to the target sequence. The tail portion comprises a rolling circle replication primer. Amplification of the signal is then performed by means of RCA. The tail portion can then be detected at the location of the target sequence.
3) Branched DNA Amplification (bDNA) (Collins et al., 1997):
The bDNA protocol includes four probe hybridization steps followed by sufficient washing after each step. First, a target-specific probe containing a small toe is added to the sample followed by a second hybridization with a pre-amplifier oligo that will bind the target-specific probe. A third hybridization with the amplifier probe that will bind the pre-amplifier is then performed. Finally, labeled probes that will hybridize with the amplifier are added. The bDNA system is based on a four-step hybridization protocol to create an amplification tree that gives sufficient signal for target probe detection. On the contrary, the probe of this disclosure is a one-molecule probe that allows target detection in a “one hybridization step” protocol.
bDNA is composed of four single-stranded oligomers that hybridize with each other to create an amplification tree. The probe of this disclosure consists of one molecule that contains a target-specific part and a signal amplification part.
A specific binding of the branched molecules in bDNA is prevented by including isobases in their sequences.
TSA is an enzyme-mediated detection method that uses horseradish peroxidase (HRP) for signal amplification. In this system, biotin-labeled probes are hybridized with the target following addition of streptavidin-HRP. Tyramide-fluorophore are deposited by HRP in the amplification reaction.
Signal amplification of target-specific probes is performed in two steps and is generated by an enzymatic process (deposition of labeled tyramide), whereas this disclosure relates to signal amplification by means of a strongly labeled signal tail sequence linked to a target-specific part.
Probes used for TSA amplification, contrary to the probes of this disclosure, do not contain a signal amplification part.
Overall drawbacks of the above-mentioned alternative methods and probes are their complex and expensive protocols, low sensitivity, low quantification possibilities, various signal amplification steps after probe hybridization, and extensive washing steps, resulting in target lost and a high background staining, which hamper their use for routine application. Thus, there is a need to design better performing probes that are capable of visualizing targets in hybridization assays and that can be used for routine application.
5) The Following Methods that may Allow Detection of Methylation changes have been Described:
5.1 Nuovo et al. (Nuovo et al., 1999) disclose methylation-specific PCR in situ hybridization (ISH). They monitored p16INK4a methylation changes in Formalin-Fixed, Paraffin-Embedded (FFPE) tissue samples. In this protocol, in situ bisulfite conversion is first performed overnight, followed by methylation-specific in situ PCR (MSP). MSP uses primers specific for detection of sequence differences between methylated versus unmethylated DNA, that result from bisulfite modification; Bisulfite modified DNA was amplified with p16INK4a gene-specific primers 5′-TTTTTAGAGGATTTGAGGGATAGG-3′ (sense, SEQ ID NO:1) and 5′-CTACCTAATTCCAATTCCCCTACA-3′ (anti-sense, SEQ ID NO:2). After amplification, in situ hybridization was performed by simultaneously adding long (>80 base pairs (bp) sized) unmethylated-specific or methylated-specific internally digoxigenin-labeled probes. Here again, PCR amplification is used for signal amplification and the efficiency of the test relies on PCR specificity followed by amplicon detection by probes. Thus, this test does not allow detection of the target at physiological levels. Moreover, because the target is amplified and the amplicons will crowd the nuclei, co-localization of multiple targets (for example, an unmethylated target and a methylated target) will be very difficult to interpret.
5.2 Larsson et al. (Larsson et al., 2004) describe padlock probes for single nucleotide polymorphisms (SNPs) detection. These are oligonucleotide probes that induce circularization of the target after hybridization to the target region. Double-stranded (dsDNA) is made accessible for padlock probe hybridization by enzymatic digestion. A combination of restriction enzymes and exonuclease enzymes (MSCI and EcoRV) is used. Following PCR amplification, labeled oligo probes are added and these recognize the amplified target. However, the efficiency of the probe hybridization is only 10% because enzymatic restriction that should expose the sense or anti-sense strand is not absolute and it is difficult to regulate. Further factors are PCR efficiency and DNA loss. This method thus combines PCR amplification for sufficient signal amplification and enzymatic restriction to allow probe access and, thus, does not allow quantification of the target at physiological levels.
5.3 Li et al. (Li et al., 2013) disclose microscopic evaluation of the methylation status at satellite repeats. This paper demonstrates the detection of the methylation status of minor and major satellite repeats using labeled Locked Nucleic Acids (LNA) probes. Probe recognition depends on cross-linking of a bipyridine-adenine derivative at the position corresponding to the methylated cytosine in the presence of osmium; therefore, the described method does not allow detection of unmethylated sequences, and so hypomethylation cannot be observed. The described method can only be used for detection of highly abundant repeats, because these small probes cannot compete with re-hybridization of the complementary strands and will not generate enough signals for microscopic evaluation of single copy genes.
In order to ensure a sufficient sensitivity for microscopic evaluation, the above-described methods must either be PCR-based (Larsson et al., 2004; Nuovo et al., 1999) or they can only detect abundant targets such as satellite repeats (Li et al., 2013). Specificity is achieved by target-specific amplification with methylation-specific primers, following in situ hybridization with >80 bp probes (Nuovo et al., 1999), cross-linking by means of a bipyridine-adenine derivative at the position corresponding to the methylated cytosine in the presence of osmium (Li et al., 2013) or ligation of padlock probes at SNP positions (Larsson et al., 2004).
The disclosure relates to a (single-stranded) probe comprising at least the following parts: a) a first part comprising a nucleotide sequence that is (semi-)complementary to a target sequence and is similar to a reverse primer, b) a second part functioning as a spacer and comprising at least one nucleotide that is not complementary to the target sequence, and c) a third part comprising a nucleotide sequence that is not complementary to the target sequence, wherein the nucleotides are composed of only three different types of nucleotides chosen from the five different types A, C, G, T or U and wherein 10% to 100% of the nucleotides are labeled.
The disclosure further relates to a probe as defined above further comprising a fourth part comprising a nucleotide sequence that is not complementary to the target sequence but is (semi-)complementary to a forward primer.
The disclosure further relates to a probe as defined above further comprising a fifth part comprising the types of nucleotides that are not chosen in the third part.
The disclosure also relates to a probe as defined above wherein the third part is made double-stranded (double-stranded probe) by PCR or by hybridization with a (semi-)complementary sequence to the signal tail.
The disclosure also relates to a process to synthesize a single-stranded) probe as defined above comprising:
The disclosure further relates to a process of synthesizing a (double-stranded) probe as defined above comprising:
The disclosure relates to a process of synthesizing a double-stranded probe by hybridization of the signal tail to a (semi-)complementary sequence to the signal tail.
The disclosure also relates to the usage of a probe as defined above to specifically detect small target sequences.
The disclosure further relates to a kit comprising a probe as defined above.
The disclosure further relates to a method of detecting methylation change-induced single nucleotide polymorphism in situ and/or to distinguish methylation heterogeneity from hemi-methylation and mono-allelic methylation in a sample taken from a patient comprising:
The disclosure relates to a method as defined above wherein the target-specific probes are probes as defined above.
The disclosure further relates to a kit comprising a target-specific probe and/or blocking probes and/or DNA protecting probes as defined above.
The disclosure finally relates to the usage of a kit as defined above to perform the method as defined above.
The disclosure includes, in the first instance, of a target-specific amplification probe called the Uniprobe Signal Amplification System (UPSAS), which targets and visualizes mutations and methylation changes in in a patient's DNA and/or detects RNA in hybridization assays (dot blot, southern blot, ISH) and its synthesis.
This disclosure thus relates to a target-specific amplification probe to detect target RNA and/or DNA sequences in hybridization assays, wherein the probe is characterized by:
In other words, this disclosure relates to a probe consisting of four major parts: a) a target-specific probe sequence with a similar sequence as the reverse primer, b) optionally, one or more spacers of at least one nucleotide (which may also include a stretch of single-type nucleotides such as an A-stretch as indicated in
The disclosure also relates to a method that allows direct targeting and visualizing of methylation changes in single copy genes in patients' DNA and RNA in situ in one step using specifically designed target-specific probes linked with a labeled compound that contains a vast number of chromogens or fluorescent dyes, to blocking probes and to DNA protecting probes.
In other words, this disclosure provides a non-PCR-based method that directly detects methylation changes in patients' DNA or RNA by incorporating a large signal-generating compound (labeled compound or signal tail) at the target-specific part of the target-specific probes rather than replicating target sequences for sufficient detection. Therefore, this disclosure allows for detection of small targets including methylation changes at physiological levels.
This disclosure further relates to the “target-specific probes,” wherein:
More specifically, UPSAS is characterized by:
The target-specific detection probe part of UPSAS is further specifically characterized by:
The “signal tail” is specifically characterized by:
The “spacers” between the signal tail and the detection probes are specifically characterized by:
An annealing site for the forward primer is included at one end (preferentially at the 3′ end) of the UPSAS probe. The forward primer annealing site is characterized by:
The forward primer annealing site is mandatory for the synthesis of sealed UPSAS in the case where the signal tail is made double-stranded by PCR to reduce aspecific binding and/or for incorporation of additional labels, but optional for the synthesis of unsealed UPSAS or in the case where the sealed probe is made double-stranded by hybridization with the seal.
Synthesis of UPSAS is performed in one (unsealed UPSAS) or two PCR steps (sealed UPSAS) (
During the probe sealing step, primer elongation is performed with only one primer (forward primer) in the presence of labeled nucleotides. After PCR2, a partially double stranded probe is generated with the detection probe still free for target recognition and binding. Elongation of forward primer is blocked at the A-repeat at the 3′ end of the target-specific probe part because probe sealing is performed with a dNTP-mix consisting of A, G, C and lacking U/T.
Synthesis of sealed UPSAS may also be performed by hybridization of unsealed UPSAS with a (semi-)complementary sequence (seal) of the signal tail.
Hence, the probe (UPSAS) of this disclosure has a double function:
This disclosure also relates to a method of detecting methylation changes and/or distinguishing methylation heterogeneity from hemi-methylation and mono-allelic methylation in situ in a sample taken from a patient comprising:
With the term “methylation changes” is meant the conversion of unmethylated cytosines into methylated cytosines and vice versa.
With the term “heterogeneity” is meant a heterogenic pattern of unmethylated and methylated target genes in the same cell/sample.
With the term “hemi-methylation” is meant methylation changes that occur at one of the two DNA strands of one allele.
With the term “mono-allelic methylation” is meant methylation changes that occur at one of the two alleles per gene.
With the term “a sample of a patient” is meant a section cut from a FFPE tissue block, a fresh frozen tissue, a cell monolayer, or a smear acquired from a patient.
With the terms “adequately dosed pepsin and/or protease K and/or HCL and/or detergent” is meant the optimal amounts of pepsin or proteinase K provided, ranging from 0.001% to 10% pepsin/proteinase K, the optimal amount of HCL provided ranging from 0.005 M HCL to 4 M HCL, an optimal amount of detergent (TRITON® X-100, TWEEN®-20) provided ranging between 0.01% and 4%.
With the terms “adequately dosed bisulfite reagents in the presence of a RNase inhibitor” is meant the optimal provided concentrations of bisulfite, NaOH and RNase inhibitor ranging from 1 to 8 M bisulfite, 0.1 M to 1 M NaOH and 1:3 to 1:10000, preferentially between 1:100 to 1:1000 RNase inhibitor, respectively.
The present disclosure relates to the “target-specific probes” to detect methylation changes characterized by:
their nature as a nucleic acid or nucleic acid analog, consisting of a small target-specific part, which is able to distinguish nucleotide polymorphisms and methylation changes, and is bound to one or more labeled compound(s), which is a nucleic acid or nucleic acid analog of which the sequence(s) shows no sequence complementarity to the human genome or to the bisulfite converted sequence of the human genome and contains 10% to 100% fluorescent-labeled nucleotides.
More specifically, “target-specific probes” are characterized by:
The disclosure further relates to blocking probes characterized by:
their nature as an unlabeled nucleic acid or nucleic acid analog, having a complementary sequence to similar sequences as the target sequence, for instance, the bisulfite-converted unmethylated counterpart sequence of the target region and having a maximal bp difference of 1 kbp and preferentially of 40 bp to the target-specific part of the target-specific probes used in the same assay.
More specifically, this disclosure relates to the blocking probes characterized by:
The present disclosure further relates to unlabeled DNA-protecting probes characterized by:
their nature as an unlabeled nucleic acid or nucleic acid analog, showing (semi-)complementarity to the sequences flanking the target sequences at the 5′ end and/or 3′ end in the DNA and/or RNA, through Watson-Crick base pairing and are used to “relax” the target sequences and enhance binding of the blocking and/or target-specific probes.
More specifically, this disclosure relates to the latter unlabeled DNA-protecting probes characterized by:
This disclosure also relates to a kit comprising UPSAS and/or blocking probes and/or unlabeled DNA-protecting probes as defined above.
This disclosure relates to the usage of the latter kit to perform the method as described above.
The term “kit” refers to any manufacture (e.g., a package or a container) comprising at least one reagent/probe as described above for performing an assay/method as described above. Positive and/or negative controls can be included in the kits to validate the activity and correct usage of reagents employed in accordance with this disclosure. The design and use of controls is standard and well within the routine capabilities of those of ordinary skill in the art. The kit can be promoted, distributed, or sold as a unit for performing the methods or usages of this disclosure. Additionally, the kits can contain a package insert describing the kit and methods/usages for its use. The term “kit” is, for example, also described in WO 2009/141359 which is hereby included by reference.
This disclosure will now be illustrated by the following, non-limiting examples.
Four different UPSAS probes corresponding to the Glutathione S-Transferase Pi 1 (GSTP1), hypermethylated regions in prostate cancer were designed and synthesized by PCR.
The four templates used for probe synthesis consist of five major parts (from 5′ end to 3′ end): 1) a sequence that is similar to the forward primer used in probe synthesis and sealing, 2) a part that consists of AGC nucleotides (template for signal tail), 3) a T-stretch of three nucleotides (this is included to stop probe sealing when probe sealing is performed with three nucleotides), 4) a spacer of nine nucleotides, and 5) a sequence that is complementary to the reverse primer and thus to the target-specific probe.
During the first PCR step, a forward primer (FP) with the same sequence as the sequence found at the 5′ end of the probe template and a reverse primer (RP) that will form the target-specific probe part of UPSAS, were used for amplification. A PCR reaction mix containing the forward primer, reverse primer, unlabeled dCTPs, dGTP and dATP, labeled dUTPs, unlabeled dUTP, Taq DNA Polymerase, PCR buffer, MgCl2, nuclease-free H2O and template, was made. Optionally, a second PCR step may be performed to (partially) seal the signal tail with a complementary sequence (“seal”).
Only the FP is used for the second PCR step; the primer is complementary to a sequence at the 3′ end of the probe. A PCR reaction mix was made containing the forward primer, unlabeled dCTPs, unlabeled dGTP, unlabeled dATP, labeled dATP, Taq DNA Polymerase, PCR buffer, MgCl2 and nuclease-free H2O. PCR2 was performed in the presence of only three nucleotides to stop elongation before the target-specific probe part, which is kept available for target binding. After the second PCR step, a target-specific probe, of which the signal tail carries a complementary sequence (“seal”) and thus prevents aspecific binding of the signal tail and carries additional signals, is generated.
Sealing of the unsealed probe may also be performed by incubating the unsealed probe with the “seal” allowing hybridization of the signal tail with the seal.
Probes were run on gel to confirm probe labeling and to estimate the amount of labels per probe. The probes contained at least 250 labels after PCR1 and 500 labels after PCR2.
Biotin-labeled GSTP1 probes were spotted on a nylon membrane prior to staining with 3,3′-Diaminobenzidine (DAB): spots stained dark brown, indicating that labels were not only incorporated in the probes as could be observed based on the molecular weight size on gel, but also gave strong signals.
3 μM formalin-fixed and paraffin-embedded (FFPE) cervical tissue sections were cut and stretched on the glass slide and deparaffinized in xylene. The sections were dehydrated two times for 5 minutes in 100% ethanol. Hereupon, the sections were incubated in 0.2 N HCl and washed with ultrapure water. The sections were treated for 28 minutes at 37° C. with porcine pepsin and washed two times for 5 minutes with ultrapure water. The sections were treated for 15 minutes with 0.1% TRITON® and washed for 3 minutes with molecular grade water afterwards. Subsequently, sections were incubated with 150 μl of a bisulfite mix (Zymo) for 4 hours. After a 15-minute desulfonation step, the samples were washed with molecular grade water and stained with hematoxylin and eosin (H&E) to evaluate conservation of the tissue morphology. Tissue morphology was evaluated by an experienced, university level pathologist, who confirmed that the morphology was kept intact.
This experiment shows that bisulfite treatment in situ and the pretreatment that should be performed prior to probe hybridization do not interfere with tissue morphology.
We compared DAPI staining between samples incubated with bisulfite for 1, 2, 3, and 4 hours. DAPI gives brighter signals when binding to double-stranded DNA than when binding to single-stranded DNA. As a control, we included a FFPE tissue slide that was not pretreated with bisulfite.
3 μM FFPE cervical tissue sections were cut and deparaffinized in xylene. The slides were dehydrated two times for 5 minutes in 100% ethanol. Hereupon, slides were incubated with RNase A (100 μg/ml RNase A in 2× Saline Sodium Citrate (SSC) for 45 minutes at room temperature (RT) and they were washed with 2×SSC (2×5 minutes). Slides were then treated with 0.2 M HCl and washed with ultrapure water. The slides were treated for 28 minutes at 37° C. with porcine pepsin and washed two times for 5 minutes with ultrapure water. The slides were treated for 15 minutes with 0.1% TRITON® and washed for 3 minutes with molecular grade water afterward. Subsequently, sections were incubated with 150 μl of a bisulfite solution (Zymo) for 1, 2, 3 and 4 hours. After a 15-minute desulfonation step, the slides were stained with DAPI.
Samples with a four-hour bisulfite incubation showed the highest reduction in DAPI brightness, indicating that the most ssDNA is generated after a four-hour bisulfite treatment.
ISH pretreatment and bisulfite conversion was performed on nine FFPE skin sections; on one set of samples, an additional HCL step was performed.
Seven μM FFPE skin sections were cut and deparaffinized in xylene. Slides were dehydrated two times for 5 minutes in 100% ethanol. Hereupon, the slides were incubated in 0.2 M HCl and washed with ultrapure water and 2×SSC. The slides were then incubated for 37 minutes in 1 M NaSCN (VWR) at 80° C. and washed with ultrapure water and 2×SSC. The slides were treated for 28 minutes at 37° C. with porcine pepsin and washed two times for 5 minutes with 2×SSC. Slides were treated for 15 minutes with 0.1% TRITON® and washed for 3 minutes with molecular grade water afterward. Subsequently, sections were incubated with 150 μl of a bisulfite solution (Zymo) for 4 hours. After a 15-minute desulfonation step, the samples were washed with molecular grade water and they were scraped off in a reaction tube containing TRIZOL®. The samples were homogenized using a tissue mixer and bisulfite-converted DNA was purified. PICOGREEN® and RIBOGREEN® were used to respectively measure ds- and ssDNA concentrations in the sample extracts. The concentration of the single-stranded product significantly increased with an increasing incubation time. PCR amplification with primers specific for the bisulfite-converted regions of two genes (ACTB and TWIST (Renard et al., 2010)) was performed; efficient qPCR amplification occurred in all cases: a higher Ct (cycle threshold) value was seen in samples treated with HCL. To confirm efficient bisulfite conversion, a set of samples (incubated with bisulfite for 4 hours) was sequenced. All samples showed 99.93% to 99.97% bisulfite conversion in the amplified regions, indicating that in situ bisulfite conversion was optimal in these samples.
Fluorescent-labeled UPSAS probes were synthesized and used to detect GSTP1 hypermethylation in breast and prostate cancer cell lines.
MCF7, LNCaP, SKBR3, BT474, PC3 and MDA-MB-231 cell lines were first tested for GSTP1 hypermethylation by MSP. In all cell lines except for MDA-MB-231, hypermethylated GSTP1 copies were detected. The amount of hypermethylated GSTP1 copies ranged between 9.7% for LNCa P and 47.1% for PC3. MDA-MB-231 was, therefore, used as a negative control for GSTP1 hypermethylation.
Four micron FFPE MCF7, LNCaP, SKBR3, BT474, PC3 and MDA-MB-231 slides were cut and deparaffinized in xylene. Slides were dehydrated in 100% ethanol. Hereupon, the slides were incubated with HCL. The slides were then washed with ultrapure water and 2×SSC. Slides were treated with porcine pepsin and washed with 2×SSC. Slides were incubated with a bisulfite solution for 3 hours at 54° C., followed by a washing step with 2×SSC. After a 15-minute desulfonation step, the slides were washed with 2×SSC. The PC3 cell line section was not treated with bisulfite but only incubated with molecular grade water. The slides were dehydrated using ethanol series (70%, 90% and 100%). After air-drying, slides were incubated overnight at 42° C. with GSTP1 methylation-specific probes. LNCaP slides were incubated with sealed or unsealed probe. Post-hybridization washes were performed in 2×SSC, 0.1×SSC and mounted for microscopic evaluation. In the majority of the cells, two dots were observed, indicating GSTP1 hypermethylation of both alleles (
Target-specific probes consist of two compartments: 1) at one end, an unlabeled target-specific part that recognizes the methylation changes (in this case, CADM1 hypermethylation (Eij sink et al., 2012; Overmeer et al., 2008), and 2) at the other end, a labeled compartment whose function is to allow signalization of the target-specific part. Both compartments form the target-specific probe. The target-specific probes are synthesized by PCR. The template that is used for the generation of the probe consists of two core sequences: 1) One sequence that is identical to the target region, and 2) one random sequence of 200 bp to 100 kbp that shows low or no sequence similarity to the human genome or to the bisulfite-converted sequence of the human genome. One or two PCR steps are used for generation of the target-specific probes. During the first PCR step, a primer that is complementary to the target-specific region is used for amplification. A reaction mix containing 500 nM primer, 100 μM unlabeled dNTPs, 100 μM labeled dNTPs, 0.3 μl Taq DNA Polymerase, PCR buffer 2 μl, 1 ng template and nuclease-free H2O was made. PCR cycling conditions are 1) Denaturation 98° C.—1 minute, 2) Cycling 1: 98° C.—20 seconds, 64° C. (sense) or 72° C. (anti-sense)—45 seconds, 72° C., 5 minutes, 20 repeats, 3) Final extension: 72° C.—1 minute, 4) Hold: 4° C., ∞ After the first PCR step, target-specific probes are generated. Optionally, a second PCR step may be performed to seal the labeled sequence with a complementary sequence. First, the probe concentration generated probe during “PCR step 1” is measured with RIBOGREEN® and 1 ng of the generated probe is used as an input for the second PCR step. One primer is used for the second PCR step, the primer is complementary to a 18 to 100 bp region at the 3′ end of the target-specific probe. A 20 μl reaction mix was made containing 500 nM primer, 200 μM unlabeled dNTPs, 0.5 [M of a ddNTP, 0.3 μl Taq DNA Polymerase, 2 μl PCR buffer, 1 ng template and nuclease-free H2O. The PCR conditions used were: 1) Denaturation 98° C.—1 minute, 2) Cycling 1: 98° C.—20 seconds, 60° C.—45 seconds, 72° C. minutes, 20 repeats, 3) Final extension: 72° C.—1 minute, 4) Hold: 4° C., co. ddNTPs are used to stop elongation before the target-specific part, which is kept available for target binding. After the second PCR step, a target-specific probe, of which the labeled component carries a complementary sequence that seals the labeled compound and thus prevents aspecific binding of the labeled compound to a patient's DNA, is recovered.
MISH was demonstrated in FFPE SiHa cell lines (cervical cancer cell line). SiHa cell lines are characterized by CADM1 gene hypermethylation, a potential biomarker for squamous cervical cancer (SCC).
Blocking probes, specific for unmethylated sequences, target-specific probes, including a labeled compound and DNA protecting probes, are designed for the CADM1 gene. These probes can be designed for any gene. Three μM FFPE SiHa cell line sections and skin sections were cut and deparaffinized in xylene. Slides were dehydrated two times for 5 minutes in 100% ethanol. Hereupon, the slides were incubated with RNase A (100 μg/ml RNase A in 2×SSC) for 45 minutes at room temperature (RT) and washed with 2×SSC (2×5 minutes). The slides were then incubated in HCl and washed with ultrapure water and 2×SSC. Slides were treated for 37 minutes with 1 M NaSCN (VWR) at 80° C. and washed for 1 minute with ultrapure water and two times for 5 minutes with 2×SSC. Slides were treated for 28 minutes at 37° C. with porcine pepsin and washed two times for 5 minutes with 2×SSC. Slides were incubated for 5 minutes with 50% formamide at 95° C. Excessive formamide was removed and the sections were incubated with a 150 μl bisulfite solution for 4 hours at 50° C. After a 15-minute desulfonation step, the slides were washed two times for 5 minutes with 2×SSC and post-fixed with 1% formaldehyde. The slides were dehydrated using an ethanol series (70%, 90% and 100%). After air-drying, slides were incubated with blocking-probes and DNA-protecting probes for 3 hours at 45° C. Post-hybridization washes were performed in 2×SSC/0.3% NP-40 at room temperature (RT), at 42° C. to 72° C. and at RT. A second incubation with CADM1 methylation-specific probes (6 nM) was performed overnight at 45° C. Post-hybridization washes were performed in 2×SSC at RT, at 42° C. to 72° C. and at RT. The slides were then incubated with Streptavidin-FITC (1:500). Unbound streptavidin was washed away with 2×SSC. Slides were counterstained with DAPI. Consequently, hypermethylated CADM1 was evaluated in situ with a fluorescence microscope. In defined conditions, SiHa cells showed predominantly two noticeable signals per cell and skin tissue cells did not show any signal, implying specific binding of CADM1 target probes (
A set of three HPV73 UPSAS probes recognizing HPV73 LI and El mRNA were designed and synthesized.
HPV73 cervical smears confirmed by qPCR using HPV73-specific primers were analyzed with the synthesized HPV73 UPSAS probes. Monolayers were fixated with paraformaldehyde (PFA), permeabilized with proteinase K and incubated with a hybridization mixture containing three HPV73 mRNA-detecting probes. Monolayers were washed and visualized with the fluorescence microscope. Individual HPV73 LI and El mRNA were adequately detected with the 40× objective (
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| Number | Date | Country | Kind |
|---|---|---|---|
| 15176744.9 | Jul 2015 | EP | regional |
This application is a national phase entry under 35 U.S.C. § 371 of International Patent Application PCT/EP2016/066510, filed Jul. 12, 2016, designating the United States of America and published in English as International Patent Publication WO 2017/009322 A1 on Jan. 19, 2017, which claims the benefit under Article 8 of the Patent Cooperation Treaty to European Patent Application Serial No. EP15176744.9, filed Jul. 15, 2015.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2016/066510 | 7/12/2016 | WO | 00 |
