US20170145486A1 - Methods for variant detection - Google Patents

Methods for variant detection Download PDF

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US20170145486A1
US20170145486A1 US15/361,280 US201615361280A US2017145486A1 US 20170145486 A1 US20170145486 A1 US 20170145486A1 US 201615361280 A US201615361280 A US 201615361280A US 2017145486 A1 US2017145486 A1 US 2017145486A1
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Prior art keywords
primer
seq
allele
polymerase
dna
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US15/361,280
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Caifu Chen
Joseph Dobosy
Pak Wah TSANG
Mark Aaron Behlke
Scott Rose
Kristin Beltz
Garrett RETTIG
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Integrated DNA Technologies Inc
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Integrated DNA Technologies Inc
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Priority to US15/361,280 priority Critical patent/US20170145486A1/en
Application filed by Integrated DNA Technologies Inc filed Critical Integrated DNA Technologies Inc
Assigned to INTEGRATED DNA TECHNOLOGIES, INC. reassignment INTEGRATED DNA TECHNOLOGIES, INC. ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: ROSE, SCOTT, BEHLKE, MARK AARON, BELTZ, Kristin, CHEN, CAIFU, DOBOSY, JOSEPH, RETTIG, GARRETT, TSANG, PAK WAH
Priority to US15/487,401 priority patent/US10886006B2/en
Priority to US15/604,204 priority patent/US20170260583A1/en
Publication of US20170145486A1 publication Critical patent/US20170145486A1/en
Assigned to INTEGRATED DNA TECHNOLOGIES, INC. reassignment INTEGRATED DNA TECHNOLOGIES, INC. ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: BAO, Yun, WANG, YU
Priority to US16/374,751 priority patent/US20190221290A1/en
Priority to US16/374,752 priority patent/US20190218611A1/en
Priority to US17/084,797 priority patent/US20210285033A1/en
Priority to US17/140,640 priority patent/US11926866B2/en
Priority to US17/226,044 priority patent/US20210395799A1/en
Priority to US18/417,992 priority patent/US20250019746A1/en
Priority to US18/816,789 priority patent/US20250137037A1/en
Priority to US18/925,638 priority patent/US20250154562A1/en
Abandoned legal-status Critical Current

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Definitions

  • the invention can be used to provide a more efficient and less error-prone method of detecting variants in DNA, such as single nucleotide polymorphisms (SNPs), multi-nucleotide polymorphisms (MNPs), and indels.
  • SNPs single nucleotide polymorphisms
  • MNPs multi-nucleotide polymorphisms
  • the invention also provides a method for performing inexpensive multi-color assays, and provides methods for visualizing multiple allele results in a two-dimensional plot.
  • RNase H2-dependent PCR (see U.S. Patent Application Publication No. US 2009/0325169 A1, incorporated by reference herein in its entirety) and standard allele-specific PCR (ASPCR) can both be utilized for mutation detection.
  • ASPCR standard allele-specific PCR
  • the DNA polymerase performs the mismatch discrimination by detection of a mismatch at or near the 3′ end of the primer. While ASPCR is sometimes successful in mismatch detection, the discrimination can be limited, due to the low mismatch detection ability of wild-type DNA polymerases.
  • the mismatch sensitivity of the RNase H2 enzyme in rhPCR allows for both sensitive detection of DNA mutations, and elimination of primer-dimer artifacts from the reaction.
  • placement of the mismatch within the primer is important. The nearer to the cleavable RNA the mismatch is located, the more discrimination is observed from the RNase H2 enzyme, and the greater the discrimination of the resulting rhPCR assay. Given the fact that most common wild-type DNA polymerases such as Taq often display low levels of mismatch detection, the polymerase cannot be solely relied upon to perform this discrimination after RNase H2 cleavage. Coupled with the repeated interrogation desired from every cycle of standard rhPCR, placing the mismatch anywhere other than immediately opposite the RNA is undesirable when utilizing these polymerases.
  • the disclosure provides assays making use of high discrimination polymerase mutants or other high mismatch discrimination polymerases to create a new assay design that can utilize mismatches located 5′ of the RNA.
  • the invention can be used to provide a more efficient and less error-prone method of detecting mutations in DNA, such as SNPs and indels.
  • the invention also provides a method for performing inexpensive multi-color assays.
  • FIG. 1 is a diagram showing two primer designs utilized in this invention.
  • Part a) is a blocked-cleavable primer designed so that the SNP of interest is 5′ of the RNA base when hybridized to a template.
  • the RNase H2 cleaves, leaving a 3′ interrogating base, which is determined to be either a match or a mismatch by the highly discriminative DNA polymerase. Thermal cycling allows for this process to continue.
  • Part b) illustrates the RNase H2 cleavage and SNP detection are identical to a), but the primer also includes a 5′ “tail” domain that includes a binding site for a probe and a universal forward primer.
  • the highly concentrated universal forward primer comes to dominate the amplification, degrading the probe when it amplifies. This cycle is repeated 25-50 ⁇ , generating the output signal.
  • This primer design may be multiplexed, allowing for one-tube multi-color assay designs.
  • FIGS. 2A and 2B are end-point fluorescence plots from the assay described in Example 1. FAM and HEX fluorescence values are plotted onto the X and Y axis.
  • FIG. 2 A is a “Universal” SNP assay for rs351855 performed with WT Taq polymerase.
  • FIG. 2B is a “Universal” SNP assay for rs351855 performed with mutant H784Q Taq polymerase, demonstrating greatly enhanced discrimination between each of the allelic variants as observed by the greater separation of the clusters in the mutant Taq case.
  • the no template controls (NTCs) squares
  • Allele 1 samples are shown as circles, allele 2 samples as diamonds, and heterozygotes as triangles. Each reaction was performed in triplicate.
  • FIGS. 3A and 3B are allelic discrimination plots with genotyping calls for rs4655751.
  • the reaction plate was cycled immediately after reaction setup (A) or held at room temperature on the benchtop for 48 hours prior to cycling (B).
  • Genotypes are tightly clustered and have good angle separation, indicating excellent allelic specificity. Each sample was assigned the correct genotyping call, and no change in performance was observed over the 48 hour hold period.
  • FIGS. 4A and 4B illustrate a side-by-side comparison of Allelic Discrimination Plots of gene CCR2, rs1799865 from a TaqMan based assay versus rhPCR.
  • Diamonds no template controls (NTCs); squares: allele 1 samples; circles: allele 2 samples; triangles: heterozygotes.
  • the rhPCR Genotyping Assay ( FIG. 4B ) achieved higher fluorescence signal compared to a traditional 5′-nuclease genotyping assay ( FIG. 4A ) while showing concordant results.
  • FIGS. 5A and 5B are Allelic Discrimination plots of tri-allelic SNP, CYP2C8 (rs72558195), using an rhPCR genotyping single tube multiplex assay on the QuantStudioTM 7 Flex platform (Thermo Fisher).
  • NTCs no template controls
  • FIG. 5B diamonds: no template controls (NTCs); squares: allele G (allele 1) samples; circles: allele C (allele 3) samples; triangles: heterozygotes.
  • FIG. 6 shows the Tri-allelic Allelic Discrimination 360plot of CYP2C8 rs72558195, using rhPCR genotyping assay with 3 allele-specific primers multiplexed in a single reaction.
  • FIG. 7 is an allelic discrimination plot illustrating the ability of the rhPCR assay to perform quantitative genotyping.
  • FIGS. 8A and 8B illustrate genotyping results and detection of allelic copy number variation that is possible with the present invention.
  • gDNA samples were tested using varying copy numbers and varying reference genotypes.
  • diamonds no template controls (NTCs); squares: allele G samples; circles: allele C samples; and triangles: heterozygotes. The resulting data correlates with the test input.
  • FIG. 9 is a schematic representation of multiplex rhPCR.
  • FIG. 10 is the resulting tape station image indicating the effectiveness of the multiplex rhPCR methods in reducing primer dimers and increasing desired amplicon yield.
  • FIG. 11 graphically represents the effectiveness of the rhPrimers in the percent of mapped reads and on-target reads.
  • the invention pertains to a methods of single-nucleotide polymorphism (SNP) discrimination utilizing blocked-cleavable rhPCR primers (see U.S. Patent Application Publication No. US 2009/0325169 A1, incorporated by reference herein in its entirety) and a DNA polymerase with high levels of mismatch discrimination.
  • the mismatch is placed at a location other than opposite the RNA base. In these situations, the majority of the discrimination comes not from the RNase H2, but from the high discrimination polymerase.
  • the use of blocked-cleavable primers with RNase H2 acts to reduce or eliminate primer-dimers and provide some increased amount of SNP or indel (insertion/deletion) discrimination ( FIG. 1 a ).
  • high discrimination is defined as any amount of discrimination over the average discrimination of WT Thermus aquaticus (Taq) polymerase.
  • Taq Thermus aquaticus
  • examples include KlenTaq® DNA polymerase (Wayne Barnes), and mutant polymerases described in U.S. Patent Application Publication No. US 2015/0191707 (incorporated by reference herein in its entirety) such as H784M, H784S, H784A and H784Q mutants.
  • a universal detection sequence(s) is added to the 5′-end of the blocked-cleavable primers.
  • the detection sequence includes a binding site for a probe, and a binding site for a universal amplification primer.
  • the primer binding site is positioned at or near the 5′-end of the final oligonucleotide and the probe binding site is positioned internally between the universal primer site and the SNP-detection primer domain.
  • Use of more than one such chimeric probe in a detection reaction wherein distinct probe binding sites are employed allows primers to be multiplexed and further allows for multiple color detection of SNPs or other genomic features.
  • Blocked-cleavable rhPCR primers reduce or eliminate primer-dimers.
  • Primer-dimers are a major problem for use of “universal” primer designs in SNP detection assays, and that limits their utility ( FIG. 1 b ). Combining a universal amplification/detection domain with a SNP primer domain in blocked-cleavable primer format overcomes this difficulty.
  • rhPCR SNP discrimination employed blocked-cleavable primers having the mismatch (SNP site) positioned opposite the single RNA base (cleavage site). While this works for many SNP targets, there are base match/mismatch pairings where sufficient discrimination is not obtained for robust base calling. Moreover, due to the high level of differential SNP discrimination observed with rhPCR, end-point detection can be difficult, especially with heterozygous target DNAs. In the proposed method, the RNA base is identical in both discriminating primers, eliminating this issue.
  • the method involves the use of blocked-cleavable primers wherein the mismatch is placed 1-2 bases 5′ of the RNA. In a further embodiment, the method involves the use of blocked-cleavable primers with three or more DNA bases 3′ of an RNA residue, and the primers are designed such that the mismatch is placed immediately 5′ of the RNA.
  • the remaining primer has a DNA residue positioned at the 3′-end exactly at the SNP site, effectively creating an ASPCR primer.
  • a high-specificity DNA polymerase can discriminate between match and mismatch with the template strand ( FIGS. 1 a and b ).
  • Native DNA polymerases such as Taq DNA polymerase, will show some level of discrimination in this primer configuration, and if the level of discrimination achieved is not sufficient for robust SNP calling in a high throughput assay format then the use of polymerases with improved template discrimination can be used.
  • mutant DNA polymerases such as those disclosed in U.S. Patent Application Publication No.
  • the invention may utilize a “tail” domain added to the 5′ end of the primer, containing a universal forward primer binding site sequence and optionally a universal probe sequence.
  • This tail would not be complementary to the template of interest, and when a probe is used, the tail would allow for inexpensive fluorescent signal detection, which could be multiplexed to allow for multiple color signal detection in qPCR ( FIG. 1 b ).
  • 1-10 cycles of initial cycling and discrimination occurs from both the RNase H2 and the DNA polymerase. After this initial pre-cycling, a highly concentrated and non-discriminatory universal forward primer comes to dominate the amplification, degrading the probe and generating the fluorescent signal when the DNA amplifies. This cycle is repeated 25-50 ⁇ , allowing for robust detection.
  • This assay design is prone to issues with primer-dimers, and the presence of the blocked-cleavable domain in the primers will suppress or eliminate these issues.
  • a forward primer is optionally used with a reverse primer, and a tail domain is added to the 5′ end of one or both of a forward and reverse primer set.
  • the tail domain comprises a universal forward primer binding site.
  • the primers can be used to hybridize and amplify a target such as a genomic sample of interest.
  • the primers would add universal priming sites to the target, and further cycles of amplification can be performed using universal primers that contain adapter sequences that enable further processing of the sample, such as the addition of P5/P7 flow cell binding sites and associated index or barcoding sequences useful in adapters for next-generation sequencing (see FIG. 9 ).
  • a high fidelity polymerase is used, which will further lower the rate of base misincorporation into the extended product and increase the accuracy of the methods of the invention.
  • CRISPR/Cas9 is a revolutionary strategy in genome editing that enables generation of targeted, double-stranded breaks (DSBs) in genomic DNA.
  • the endonuclease activity is followed by an endogenous repair process that leads to some frequency of insertions/deletions/substitutions in wild-type DNA at the target locus which gives the resultant genome editing.
  • RNase H-cleavable primers have been designed to flank edited loci in order to 1) generate locus-specific amplicons with universal tails, and 2) be subsequently amplified with indexed P5/P7 universal primers for next-generation sequencing.
  • this strategy resulted in reliable, locus-specific amplification which captures CRISPR/Cas9 editing events in a high-throughput and reproducible manner.
  • the key finding is that the overall targeted editing by this NGS-based method was determined to be 95%; whereas, previous enzymatic strategies suggested overall editing from the same samples was approximately 55% at the intended target site.
  • primers were designed to amplify off-target locations of genomic editing based on in silico predictions by internal bioinformatics tools.
  • RNase H2 can cleave at positions containing one or more RNA bases, at 2′-modified nucleosides such as 2′-fluoronucleosides.
  • the primers can also contain nuclease resistant linkages such as phosphorothioate, phosphorodithioate, or methylphosphonate.
  • “Complement” or “complementary” as used herein means a nucleic acid, and can mean Watson-Crick (e.g., A-T/U and C-G) or Hoogsteen base pairing between nucleotides or nucleotide analogs of nucleic acid molecules.
  • Fluorophore or “fluorescent label” refers to compounds with a fluorescent emission maximum between about 350 and 900 nm.
  • Hybridization refers to the formation of a duplex structure by two single-stranded nucleic acids due to complementary base pairing. Hybridization can occur between fully complementary nucleic acid strands or between “substantially complementary” nucleic acid strands that contain minor regions of mismatch. “Identical” sequences refers to sequences of the exact same sequence or sequences similar enough to act in the same manner for the purpose of signal generation or hybridizing to complementary nucleic acid sequences. “Primer dimers” refers to the hybridization of two oligonucleotide primers. “Stringent hybridization conditions” as used herein means conditions under which hybridization of fully complementary nucleic acid strands is strongly preferred.
  • a first nucleic acid sequence for example, a primer
  • a second nucleic acid sequence for example, a target sequence
  • Stringent conditions are sequence-dependent and will be different in different circumstances.
  • Stringent conditions can be selected to be about 5-10° C. lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength pH.
  • Tm can be the temperature (under defined ionic strength, pH, and nucleic concentration) at which 50% of an oligonucleotide complementary to a target hybridize to the target sequence at equilibrium (as the target sequences are present in excess, at Tm, 50% of the probes are occupied at equilibrium).
  • Stringent conditions can be those in which the salt concentration is less than about 1.0 M sodium ion, such as about 0.01-1.0 M sodium ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30° C. for short probes (e.g., about 10-50 nucleotides) and at least about 60° C. for long probes (e.g., greater than about 50 nucleotides). Stringent conditions can also be achieved with the addition of destabilizing agents such as formamide. For selective or specific hybridization, a positive signal can be at least 2 to 10 times background hybridization.
  • Exemplary stringent hybridization conditions include the following: 50% formamide, 5 ⁇ SSC, and 1% SDS, incubating at 42° C., or, 5 ⁇ SSC, 1% SDS, incubating at 65° C., with wash in 0.2 ⁇ SSC, and 0.1% SDS at 65° C.
  • nucleic acid refers to at least two nucleotides covalently linked together.
  • the depiction of a single strand also defines the sequence of the complementary strand.
  • a nucleic acid also encompasses the complementary strand of a depicted single strand.
  • Many variants of a nucleic acid can be used for the same purpose as a given nucleic acid.
  • a nucleic acid also encompasses substantially identical nucleic acids and complements thereof.
  • a single strand provides a probe that can hybridize to a target sequence under stringent hybridization conditions.
  • a nucleic acid also encompasses a probe that hybridizes under stringent hybridization conditions.
  • Nucleic acids can be single stranded or double stranded, or can contain portions of both double stranded and single stranded sequences.
  • the nucleic acid can be DNA, both genomic and cDNA, RNA, or a hybrid, where the nucleic acid can contain combinations of deoxyribo- and ribonucleotides, and combinations of bases including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine hypoxanthine, isocytosine and isoguanine.
  • Nucleic acids can be obtained by chemical synthesis methods or by recombinant methods.
  • a particular nucleic acid sequence can encompass conservatively modified variants thereof (e.g., codon substitutions), alleles, orthologs, single nucleotide polymorphisms (SNPs), and complementary sequences as well as the sequence explicitly indicated.
  • PCR Polymerase Chain Reaction
  • the reaction typically involves the use of two synthetic oligonucleotide primers, which are complementary to nucleotide sequences in the substrate DNA which are separated by a short distance of a few hundred to a few thousand base pairs, and the use of a thermostable DNA polymerase.
  • the chain reaction consists of a series of 10 to 40 cycles. In each cycle, the substrate DNA is first denatured at high temperature. After cooling down, synthetic primers which are present in vast excess, hybridize to the substrate DNA to form double-stranded structures along complementary nucleotide sequences.
  • the primer-substrate DNA complexes will then serve as initiation sites for a DNA synthesis reaction catalyzed by a DNA polymerase, resulting in the synthesis of a new DNA strand complementary to the substrate DNA strand.
  • the synthesis process is repeated with each additional cycle, creating an amplified product of the substrate DNA.
  • Primer refers to an oligonucleotide capable of acting as a point of initiation for DNA synthesis under suitable conditions. Suitable conditions include those in which hybridization of the oligonucleotide to a template nucleic acid occurs, and synthesis or amplification of the target sequence occurs, in the presence of four different nucleoside triphosphates and an agent for extension (e.g., a DNA polymerase) in an appropriate buffer and at a suitable temperature.
  • a DNA polymerase an agent for extension
  • Probe and “fluorescent generation probe” are synonymous and refer to either a) a sequence-specific oligonucleotide having an attached fluorophore and/or a quencher, and optionally a minor groove binder or b) a DNA binding reagent, such as, but not limited to, SYBR® Green dye.
  • Quencher refers to a molecule or part of a compound, which is capable of reducing the emission from a fluorescent donor when attached to or in proximity to the donor. Quenching may occur by any of several mechanisms including fluorescence resonance energy transfer, photo-induced electron transfer, paramagnetic enhancement of intersystem crossing, Dexter exchange coupling, and exciton coupling such as the formation of dark complexes.
  • RNase H PCR refers to a PCR reaction which utilizes “blocked” oligonucleotide primers and an RNase H enzyme.
  • “Blocked” primers contain at least one chemical moiety (such as, but not limited to, a ribonucleic acid residue) bound to the primer or other oligonucleotide, such that hybridization of the blocked primer to the template nucleic acid occurs, without amplification of the nucleic acid by the DNA polymerase.
  • the blocked primer hybridizes to the template or target nucleic acid, the chemical moiety is removed by cleavage by an RNase H enzyme, which is activated at a high temperature (e.g., 50° C. or greater). Following RNase H cleavage, amplification of the target DNA can occur.
  • the 3′ end of a blocked primer can comprise the moiety rDDDDMx, wherein relative to the target nucleic acid sequence, “r” is an RNA residue, “D” is a complementary DNA residue, “M” is a mismatched DNA residue, and “x” is a C3 spacer.
  • a C3 spacer is a short 3-carbon chain attached to the terminal 3′ hydroxyl group of the oligonucleotide, which further inhibits the DNA polymerase from binding before cleavage of the RNA residue.
  • RNase H-dependent PCR reactions are performed using an RNase H enzyme obtained or derived from the hyperthermophilic archaeon Pyrococcus abyssi (P.a.), such as RNase H2.
  • RNase H enzyme employed in the methods described herein desirably is obtained or derived from Pyrococcus abyssi , preferably an RNase H2 obtained or derived from Pyrococcus abyssi .
  • the RNase H enzyme employed in the methods described herein can be obtained or derived from other species, for example, Pyrococcus furiosis, Pyrococcus horikoshii, Thermococcus kodakarensis , or Thermococcus litoralis.
  • This example demonstrates an enhanced rhPCR assay that utilizes a highly discriminatory DNA polymerase and RNase H2 for discrimination
  • rhPrimers and standard allele-specific primers were designed against rs113488022, the V600E mutation in the human BRAF gene. These primers were tested in PCR and rhPCR with WT or H784Q mutant Taq polymerase. Primers utilized in these assays were as shown in Table 1 (SEQ ID NOs: 1-7).
  • ZEN internal ZEN TM quencher (IDT, Coralville, IA)
  • FAM 6-carboxyfluorescein
  • IBFQ Iowa Black ® FQ (fluorescence quencher, IDT, Coralville, IA)
  • x C3 propanediol spacer block
  • reaction volumes were used in these assays.
  • 5 ⁇ L of 2 ⁇ Integrated DNA Technologies (IDT) (Coralville, Iowa) rhPCR genotyping master mix (containing dNTPs, H784Q mutant or WT Taq DNA polymerase, stabilizers, and MgCl 2 ) was combined with 200 nM (2 pmol) of either of the allelic primers.
  • 200 nM (2 pmol) of the probe, as well as 200 nM (5 pmol) of the reverse primer were also added.
  • RNase H2 and 1000 copies of synthetic gBlockTM (Integrated DNA Technologies, Coralville, Iowa) template 1000 copies Allele 1, 500 copies allele 1+500 copies allele 2 (heterozygote), or 1000 copies Allele 2 (for gBlockTM sequences, see Table 2, SEQ ID NOs: 8-9) were added to the reaction mix.
  • the reaction was thermocycled on a Bio-RadTM CFX384TM Real-time system. Cycling conditions were as follows: 953:00 ⁇ (950:10 ⁇ 650:30) ⁇ 65 cycles. Each reaction was performed in triplicate.
  • the following example demonstrates an enhanced rhPCR assay that utilizes a highly discriminatory DNA polymerase and RNase H2 for discrimination.
  • rhPrimers and standard allele-specific primers were designed against rs113488022, the V600E mutation in the human BRAF gene. These primers were tested in PCR and rhPCR with H784Q mutant Taq polymerase. Primers utilized in these assays were as shown in Table 4 (SEQ ID NOs: 1, 4 and 10-12).
  • RNase H2 and 5e4 copies of synthetic gBlockTM (Integrated DNA Technologies, Coralville, Iowa) template (1e5 copies Allele 1, 5e4 copies allele 1+5e4 copies allele 2 (heterozygote), or 1e5 copies Allele 2 (for gBlockTM sequences, see Table 2, SEQ ID NOs: 8-9) were added to the reaction mix.
  • the reaction was thermocycled on a Bio-RadTM CFX384TM Real-time system. Cycling conditions were as follows: 95 3:00 ⁇ (95 0:10 ⁇ 65 0:30 ) ⁇ 65 cycles. Each reaction was performed in triplicate.
  • the following example illustrates the heightened reliability of universal assays using a DNA polymerase with a high mismatch discrimination.
  • RNase H2 and 1000 copies of synthetic gBlockTM (Integrated DNA Technologies, Coralville, Iowa) template (1000 copies Allele 1, 500 copies allele 1+500 copies allele 2 (heterozygote), or 1000 copies Allele 2 (for gBlockTM sequences, see Table 7, SEQ ID NOs: 19-20) were added to the reaction mix.
  • the reaction was thermocycled on a Bio-RadTM CFX384TM Real-time system. Cycling conditions were as follows: 95 3:00 ⁇ (95 0:10 ⁇ 60 0:30 ) ⁇ 3 cycles ⁇ (95 0:10 ⁇ 65 0:30 ) ⁇ 65 cycles. Each reaction was performed in triplicate. Fluorescence reads were taken after a total of 50 cycles were completed. Fluorescence values were plotted on the FAM and HEX axis, and results are shown in FIGS. 2 a and 2 b .
  • reaction volumes were used in these assays.
  • 5 ⁇ L of 2 ⁇ Integrated DNA Technologies (IDT) (Coralville, Iowa) rhPCR genotyping master mix (containing dNTPs, H784Q mutant or WT Taq DNA polymerase, stabilizers, and MgCl 2 ) was combined with 200 nM (2 pmol) of either of the allelic primers, or the non-discriminatory forward primer.
  • 200 nM (2 pmol) of the probe, as well as 200 nM (5 pmol) of the reverse primer were also added.
  • the reaction was thermocycled on a Bio-RadTM CFX384TM Real-time system. Cycling conditions were as follows: 95 3:00 ⁇ (95 0:10 ⁇ 60 0:30 ) ⁇ 65 cycles. Each reaction was performed in triplicate.
  • This example demonstrates successful allelic discrimination with the use of a universal rhPCR genotyping assay and Integrated DNA Technologies (IDT) (Coralville, Iowa) rhPCR genotyping master mix, and the robust stability of the reaction components.
  • IDT Integrated DNA Technologies
  • universal primers were designed against rs4657751, a SNP located on the human Chromosome 1 (See Table 11, SEQ ID NOs: 14, 21-25).
  • rhPCR assay primers 150 nM of rs4657751 Allele Specific Primer 1 (SEQ ID NO: 23), 150 nM of rs4657751 Allele Specific Primer 2 (SEQ ID NO: 24), and 500 nM rs4657751 Locus Specific Primer (SEQ ID NO: 25).
  • Reactions contained universal reporter oligos at the following concentrations: 250 nM of universal FAM probe (SEQ ID NO: 14), 450 nM of universal Yakima Yellow® (SEQ ID NO: 22) probe, and 1000 nM of universal forward primer (SEQ ID NO: 21), and 5 ⁇ L of 2 ⁇ Integrated DNA Technologies (IDT) (Coralville, Iowa) rhPCR genotyping master mix (containing dNTPs, a mutant H784Q Taq polymerase (see Behlke, et al. U.S. 2015/0191707), chemically modified Pyrococcus abyssi RNase H2 (See Walder et al. UA20130288245A1), stabilizers, and MgCl 2 ).
  • IDTT Integrated DNA Technologies
  • gBlocks® Gene Fragments (Integrated DNA Technologies, Inc., Coralville, Iowa) containing either allele of the rs4657751 SNP were utilized as the source of template DNA (See Table 12, SEQ ID NOs: 26 and 27).
  • Each well contained template representing one of three possible genotypes: allele 1 homozygote (1000 copies rs4657751 Allele 1 gBlock® template (SEQ ID NO: 26)), allele 2 homozygote (1000 copies rs4657751 Allele 2 gBlock® template (SEQ ID NO: 27)), or heterozygote (mix of 500 copies of rs4657751 Allele 1 gBlock® template (SEQ ID NO: 26) and 500 copies of rs4657751 Allele 2 gBlock® template (SEQ ID NO: 27)).
  • Template or water for the no template control (NTC) reactions were added into three replicate wells of two individual plates. The reactions underwent the following cycling protocol: 95° C. for 10 minutes,
  • LNA residues are designated with a carboxyfluorescein
  • Yak Yakima Yellow (3-(5,6,4′,7′-tetrachloro-5′-methyl-3′,6′-dipivaloylfluorescein-2-yl))
  • IBFQ Iowa Black FQ (fluorescence quencher)
  • x C3 propanediol spacer block.
  • the following example compares the performance of the genotyping methods of the present invention versus traditional 5′ nuclease genotyping assay methods (TaqmanTM).
  • Thers1799865 SNP in the CCR2 gene was selected, and rhPCR genotyping primers as well as an rs1799865 5′ nuclease assay (Thermo-Fisher (Waltham, Mass.)), were designed and obtained. Sequences for the rs1799865 rhPCR genomic SNP assay are shown in Table 14 (SEQ ID NOs: 14, 21, 22, and 28-30). Thermo-Fisher 5′ nuclease primer/probe (TaqmanTM) sequences are not published, and therefore are not included in this document.
  • Reactions were performed in 10 ⁇ L volumes, containing 10 ng Coriell genomic DNA (Camden, N.J.), 250 nM of universal FAM probe (SEQ ID NO: 14), 450 nM of universal Yakima Yellow® (SEQ ID NO: 22) probe, 1000 nM of universal forward primer (SEQ ID NO: 21), 150 nM of the two allele-specific forward primers (SEQ ID NOs: 28 and 29), 500 nM of the reverse primer (SEQ ID NO: 30), and 5 ⁇ L of 2 ⁇ Integrated DNA Technologies (IDT) (Coralville, Iowa) rhPCR genotyping master mix (containing dNTPs, a mutant H784Q Taq polymerase (see Behlke, et al. U.S. 2015/0191707), chemically modified Pyrococcus abyssi RNase H2 (See Walder et al. UA20130288245A1), stabilizers, and MgCl 2 ).
  • IDTT
  • PCR was performed on Life Technologies (Carlsbad, Calif.) QuantStudioTM 7 Flex real-time PCR instrument using the following cycling conditions: 10 mins at 95° C. followed by 50 cycles of 95° C. for 10 seconds and 60° C. for 45 seconds. End-point analysis of each of the plates was performed after 45 cycles with the QuantStudioTM Real-Time PCR Software v1.3 (Carlsbad, Calif.).
  • FAM 6-carboxyfluorescein
  • Yak Yakima Yellow (3-(5,6,4′,7′-tetrachloro-5′-methyl-3′,6′-dipivaloylfluorescein-2-yl))
  • IBFQ Iowa Black FQ (fluorescence quencher)
  • x C3 propanediol spacer block.
  • FIGS. 4A and 4B show a side-by-side comparison of the resulting allelic discrimination plots.
  • the rhPCR Genotyping Assay ( FIG. 4B ) achieved higher fluorescence signal compared to a traditional 5′-nuclease genotyping assay ( FIG. 4A ) while showing concordant results.
  • the higher signal and minimal non-specific amplification from NTC in the rhPCR assay allow better cluster separation and accurate genotype calls.
  • the following example illustrates the present methods allowing for detection and analysis of tri-allelic SNP.
  • the rs72558195 SNP is present in the CYP2C8 gene, and has three potential genotypes. This SNP was selected for analysis with the rhPCR genotyping system.
  • Reactions contained universal reporter oligos at the following concentrations: 250 nM of universal FAM probe (SEQ ID NO: 14), 450 nM of universal Yakima Yellow® (SEQ ID NO: 22) probe, and 1000 nM of universal forward primer (SEQ ID NO: 21), 50 nM ROX internal standard, and 5 ⁇ L of 2 ⁇ Integrated DNA Technologies (IDT) (Coralville, Iowa) rhPCR genotyping master mix (containing dNTPs, a mutant H784Q Taq polymerase (see Behlke, et al. U.S. 2015/0191707), chemically modified Pyrococcus abyssi RNase H2 (See Walder et al. UA20130288245A1), stabilizers, and MgCl 2 ).
  • IDTT Integrated DNA Technologies
  • LNA residues are designated with a +. Location of potential mismatch in underlined.
  • FAM 6-carboxyfluorescein
  • Yak Yakima Yellow (3-(5,6,4′,7′-tetrachloro-5′-methyl-3′,6′-dipivaloylfluorescein-2-yl))
  • IBFQ Iowa Black FQ (fluorescence quencher)
  • x C3 propanediol spacer block.
  • gBlocks® Gene Fragments (Integrated DNA Technologies, Inc., Coralville, Iowa) containing alleles of the rs72558195 SNP were utilized as the source of template DNA (See Table 17, SEQ ID NOs: 35, 36 and 37).
  • heterozygote (mix of 500 copies of rs72558195 Allele 1 gBlock® template (SEQ ID NO: 35) and 500 copies of rs72558195 Allele 3 gBlock® template (SEQ ID NO: 37)).
  • Template or water for the no template control (NTC) reactions were added into three replicate wells of two individual plates. The reactions underwent the following cycling protocol: 95° C. for 10 minutes, then 45 cycles of 95° C. for 10 seconds and 60° C. for 45 seconds.
  • a Tri-allelic AD 360plot was designed for illustrating allelic discrimination. Fluorescence signal ( ⁇ Rn) from the last PCR cycle of each dye was normalized across the three dyes from the same well. Angle and distance of data point from the origin is calculated using formula below:
  • FIG. 5B shows the Tri-allelic Allelic Discrimination 360plot of rs72558195, using rhPCR genotyping assay with 3 allele-specific primers multiplexed in a single reaction.
  • the distance of data points from origin indicated the signal strength of dyes and the wide angle separation between data clusters indicated specificity of multiplex assay.
  • NTC in the center of the plot indicated no primer dimers or non-specific amplification.
  • the specificity of multiplex assay is achieved by the selectivity of RNase H2 and the mutant Taq DNA polymerase as used in the previous examples. This AD 360plot will also enable auto-calling capability by genotyping software.
  • a 360plot could be implemented for tetra-allelic, penta-allelic or hexa-allelic visualization. Therefore, visualization is possible for positions that could have multiple bases as well as potential deletions. The distance from origin remains unchanged for each calculation, and the angle formulas would be:
  • the following example illustrates the capability of the methods of the present invention to provide quantitative SNP genotyping, allowing for determination of the copy numbers of different alleles.
  • an assay was designed against rs1135840, a SNP in the human CYP2D6 gene. This gene can be present in multiple copies, and the number of copies with the rs1135840 SNP appears to affect drug metabolism (rapid metabolism of the drug Debrisoquine).
  • Reactions were performed in 10 ⁇ L volumes, containing a total of 1500 copies of template at the ratios shown (10:0, 9:1, 8:2, 7:3, 6:4, 5:5, 4:6, 3:7, 2:8, 1:9, and 0:10), 250 nM of universal FAM probe (SEQ ID NO: 14), 450 nM of universal Yakima Yellow® (SEQ ID NO: 22) probe, 1000 nM of universal forward primer (SEQ ID NO: 21), 150 nM of the two allele-specific forward primers, 500 nM of the reverse primer, and 5 ⁇ L of 2 ⁇ Integrated DNA Technologies (IDT) (Coralville, Iowa) rhPCR genotyping master mix (containing dNTPs, a mutant H784Q Taq polymerase (see Behlke, et al. U.S. 2015/0191707), chemically modified Pyrococcus abyssi RNase H2 (See Walder et al. UA20130288245A1)
  • PCR was performed on Life Technologies (Carlsbad, Calif.) QuantStudioTM 7 Flex real-time PCR instrument using the following cycling conditions: 10 mins at 95° C. followed by 45 cycles of 95° C. for 10 seconds and 60° C. for 45 seconds. End-point analysis of each of the plates was performed after 45 cycles with software provided by the respective companies (Bio-Rad CFX Manager 3.1 software (Bio-Rad, Hercules, Calif.) and QuantStudioTM Real-Time PCR Software v1.3 (Carlsbad, Calif.)).
  • FAM 6-carboxyfluorescein
  • Yak Yakima Yellow (3-(5,6,4′,7′-tetrachloro-5′-methyl-3′,6′-dipivaloylfluorescein-2-yl))
  • IBFQ Iowa Black FQ (fluorescence quencher)
  • x C3 propanediol spacer block.
  • the resulting data is illustrated in FIG. 7 .
  • the spread of each of the sample mixes is sufficient for the determination of the number of copies of each template.
  • Each individual assay also contained 50 nM ROX normalizer oligo, 250 nM of universal FAM probe (SEQ ID NO: 14), 450 nM of universal Yakima Yellow® (SEQ ID NO: 22) probe, 1000 nM of universal forward primer (SEQ ID NO: 21), 150 nM of the two allele-specific forward primers (SEQ ID NO: 38 and 39), 500 nM of the reverse primer (SEQ ID NO: 40), and 5 ⁇ L of 2 ⁇ Integrated DNA Technologies (IDT) (Coralville, Iowa) rhPCR genotyping master mix (containing dNTPs, a mutant H784Q Taq polymerase (see Behlke, et al. U.S.
  • FAM 6-carboxyfluorescein
  • Yak Yakima Yellow (3-(5,6,4′,7′-tetrachloro-5′-methyl-3′,6′-dipivaloylfluorescein-2-yl)
  • IBFQ Iowa Black FQ (fluorescence quencher).
  • Quantitative PCR was performed on Life Technologies (Carlsbad, Calif.) QuantStudioTM 7 Flex real-time PCR instrument using the following cycling conditions: 10 mins at 95° C. followed by 45 cycles of 95° C. for 10 seconds and 60° C. for 45 seconds. End-point analysis of each of the plates was performed after 45 cycles with the QuantStudioTM Real-Time PCR Software v1.3 (Carlsbad, Calif.) software provided by the company.
  • Copy number was determined by the following method. For each sample shown to be a homozygote, ⁇ Cq (RNase P Cq ⁇ rs1135840 assay Cq) was calculated for each sample. For samples shown to be heterozygotes, ⁇ Cq was calculated for both alleles (RNase P Cq ⁇ rs1135840 assay 1 Cq and RNase P Cq ⁇ rs1135840 assay 2 Cq). Next, ⁇ Cq ( ⁇ Cq ⁇ mean ⁇ Cq for known 2 copy control DNA samples) was calculated for each allele. This correction allowed for normalization against amplification differences between the SNP assay and the RNase P assay. Finally, the following equation was used to calculate copy number for each allele:
  • the resulting end-point data is shown in FIG. 8A and calculated copy numbers are shown in FIG. 8B .
  • the genotypes determined in FIG. 8A (homozygotes allele 1, Homozygotes allele 2, or heterozygote) all matched the known genotypes, and allowed correct calculation of the copy number.
  • the established reference copy number of the individual samples is shown under each result. In each case, the copy number determined by the assay correctly determined the genotype and copy number of the input DNA.
  • the following example demonstrates that a variation of an rhPCR probe can be used for multiplexed rhPCR.
  • the assay schematic is provided in FIG. 9 .
  • 5′ tailed target-specific rhPrimers are used.
  • the 5′ tails upon incorporation into the amplicon contain binding sites for a second round of PCR with different primers (blocked or unblocked) to add application specific sequences.
  • this system can be used for amplification enrichment for next generation sequencing.
  • 5′ tailed rhPCR primers contain read 1/read 2 primer sequences.
  • the second round of PCR adds adapter sequences such as the P5/P7 series for Illumina® based sequencing platforms or other adaptors, including ones containing barcodes/unique molecular identifiers. This approach allows for adding any additional sequences onto the amplicon necessary for input into any NGS platform type.
  • two primers sets including one containing a 96-plex set of 5′ tailed rhPrimers, and one containing 96 DNA “standard” 5′ tailed PCR primers were designed using an IDT algorithm.
  • the two primer sets differed only in that the rhPrimers contained an internal cleavable RNA base and a blocking group on the 3′ end. Once the blocking group was removed by RNase H2 cleavage, the primer sequences become identical.
  • the first round of PCR reactions contained the 96 plex at 10 nM of each blocked target specific primer, 10 ng of NA12878 human genomic DNA (Coriell Institute for Medical Research, Camden, N.J.), 200 mU of chemically modified Pyrococcus abyssi RNase H2 (See Walder et al. UA20130288245A1) (IDT, Coralville, Iowa) and 1 ⁇ KAPA 2G HotStart Fast Ready MixTM (Kapa Biosystems, Wilmington, Mass.).
  • the thermal cycling profile was 10 mins at 95° C. followed by 8 cycles of 95° C. for 15 seconds and 60° C. for 4 minutes, and a final 99° C. finishing step for 15 minutes.
  • Reactions were cleaned up with a 2 ⁇ AMPureTM XP beads (Beckman Coulter, Brea, Calif.). Briefly, 100 ⁇ L AMPureTM SPRI beads were added to each PCR well, incubated for 5 minutes at room temperature and collected for 5 minutes at room temperature on plate magnet (DynaMagTM (Thermo-Fisher, (Watherham, Mass.) 96-well plate side-magnet). Beads were washed twice with 80% ethanol, and allowed to dry for 3 minutes at room temperature. Samples were eluted in 22 ⁇ L of TE at pH 8.0.
  • the second round of PCR was set up using 20 ⁇ L of the cleaned up first round PCR products, universal PCR-50F and PCR-47R primers (See table 18, SEQ ID NOs: 44 and 45) at 2 uM and 1 ⁇ KAPA 2G HotStart Fast Ready MixTM (KAPA Biosystems, Wilmington, Mass.). Reactions were cycled for 45 seconds at 98° C. followed by 20 cycles of 98° C. for 15 seconds, 60° C. for 30 seconds, and 72° C. for 30 seconds. A final 1 minute 72° C. polishing step finished the reaction. Samples were cleaned up again with 0.8 ⁇ AMPureTM beads.
  • AMPureTM SPRI beads were added the second PCR wells, incubated for 5 minutes at room temperature and collected for 5 minutes at room temperature on plate magnet (DynaMagTM (Thermo-Fisher, (Watherham, Mass.) 96-well plate side-magnet). Beads were washed twice with 80% ethanol, and allowed to dry for 3 minutes at room temperature. Samples were eluted in 22 ⁇ L of TE at pH 8.0, and 20 ⁇ L was transferred to a new tube.
  • DynaMagTM Thermo-Fisher, (Watherham, Mass.) 96-well plate side-magnet
  • FIG. 10 shows the results from the Agilent® Tape Station.
  • the primer dimer product was the most significant product produced using standard DNA primers in the presence of DNA template, with only a small amount of full length expected product. In the absence of template, the primer dimer product was the major component of the reaction. In the case of the blocked rhPCR primers, the vast majority of the material was the desired PCR products, with little primer dimer observed. In the absence of template, there is no primer dimer present, contrasting with the overwhelming abundance of primer dimer observed in the no template lane of the unblocked DNA primers. Quantitation of the product versus primer dimer bands show that mass ratio of product to primer dimer for the unblocked DNA primers was 0.6. The mass ratio for the rhPCR primers was 6.3.
  • FIG. 11 summarizes two key sequencing metrics. The first is the percent of mapped reads from the sequencing data. The rhPCR reactions gave a percentage of reads mapped to the human genome at 85%, whereas the non-blocked DNA primers on give a mapped read percentage of less than 20. A second metric, the percentage of on-target reads, is almost 95% when using rhPCR primers, but less than 85% when the non-blocked primers are used in the multiplex. These results clearly demonstrate the utility of using rhPCR in multiplexing, where a large increase of the desired material is seen, and a vast reduction in undesired side products is observed. The differences mean less unwanted sequencing reads, and the depth of coverage of desired sequences is higher.

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