RNase H2 PCR: A New Molecular Technology
Dr Joseph Dobosy
Senior Research Scientist
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INTEGRATED DNA TECHNOLOGIES
Agenda
• How RNase H2 PCR (rhPCR) works
• Why use Pyrococcus abyssi RNase H2
• Two rhPrimer designs—what they are and when to use them• GEN1 rhPrimer design
• GEN2 rhPrimer design
• Applications for each generation of rhPrimers
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INTEGRATED DNA TECHNOLOGIES
Critical Features of Type II RNase H Enzymes
• Cleaves only double-stranded RNA/DNA heteroduplex at the 5′ DNA-RNA linkage
• Cleavage products have a 3′-OH and 5′-Phos 3′-OH is extendable by polymerases
rhPCR technology exploits this feature of the enzyme
• Only requires a single RNA residue to cleave
Note: Single-stranded ribonucleases, like RNase A, leave a 3′-Phos and 5′-OH, which are non-extendable
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INTEGRATED DNA TECHNOLOGIES
RNase H2 PCR: How Does It Work?
• Primer deblocking is required for PCR, which in turn required that primers be annealed to the target DNA sequence.
• The enzymatic deblocking cleavage event is sensitive to base mismatch, which confers added specificity to the ensuing PCR.
• Primer-dimer formation is highly reduced.
• Note that this is biased amplification, not biased signal generation.
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INTEGRATED DNA TECHNOLOGIES
Biased Amplification Versus Biased Signal Generation
During PCR, signal is made when the probe is degraded.
If multiple targets are present (which occurs in rare allelereactions), signal generation occurs only from the targetedallele and intensity is lowered proportionally when otheramplification targets are present.
F
F QSNP
Biased signal generation (5′ Nuclease Assays) Biased amplification (rhPCR)
rddddmx
SNP
Only the sequence of interest (red) is amplified, meaning thata single target can be identified in a background containing ahigh amount of nearly identical DNA (blue).
Detection of rare alleles can occur in a background of>1000-fold higher untargeted DNA
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INTEGRATED DNA TECHNOLOGIES
Differences Between rhPCR and Probe-Based Genotyping
rhPCR Probe-Based Genotyping
Biased amplification of alleles Biased signal generation and detection
Need to test each allele in a separate tube Can multiplex in a single tube (single reaction)
Good for rare allele Equal amplification of both alleles
Inexpensive (uses intercalating dyes) Probes are expensive up front
Good for reducing primer-dimers in highly multiplexed situations (<20 primer pairs)
Highly multiplexed reactions can cause primer-dimers to form
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INTEGRATED DNA TECHNOLOGIES
Overview: Why Use Pyrococcus abyssi RNase H2
• Very thermostable
o ONLY highly active at high temperature
o Can survive thermocycling, which most other RNase H2 enzymes cannot
• Inactive at low temperature—“Hot start” is achieved without need for a modified “hot start” polymerase
• Active across a wide range of magnesium concentrations, including concentrations commonly used in PCR
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INTEGRATED DNA TECHNOLOGIES
Active During Thermal Cycling
P. abyssi RNase H2 can be incubated at 95oC for >45 minutes with little loss of activity.
This thermostable enzyme will survive thermal cycling (e.g., PCR).
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Active at Elevated Temperatures Compatible with PCR
5’ 32P-CTCGTGAGGTGATGcAGGAGATGGGAGGCG 3’
3’ GAGCACTCCACTACGTCCTCTACCCTCCGC 5’
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INTEGRATED DNA TECHNOLOGIES
Active Across a Broad Range of Mg++ Levels
Usually 3 mM Mg++
is recommended
5’ 32P-CTCGTGAGGTGATGcAGGAGATGGGAGGCG 3’
3’ GAGCACTCCACTACGTCCTCTACCCTCCGC 5’
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INTEGRATED DNA TECHNOLOGIES
Detergent is Essential for Robust P. abyssi RNase H2 Activity
0.1% Triton X-100 is present in the dilution and storage buffer
5’-CTCGTGAGGTGATGcAGGAGATGGGAGGCG-3’
3’-GAGCACTCCACTACGTCCTCTACCCTCCGC-5’
0
10
20
30
40
50
60
70
80
90
100
Triton-X-100 Tween-20 Tween-80 Ctab N-Lauroyl sarcosinate
Perc
en
t C
leaved
0.0001
0.001
0.01
Percent detergent
in reaction
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Two rhPrimer Designs are Available
1st gen—rDDDDmx—good for general purposeGEN1 rhPrimers are most appropriate for standard genotyping applications and for multiplexed amplification
2nd gen—rDxxDm—best specificity, but at a cost: higher enzyme amounts and specific titration for applications is needed
GEN2 rhPrimers are most appropriate for rare-allele detection or for applications where extremely high fidelity of template amplification is desired
r = RNA base; D = DNA base; m = mismatched base; x = C3 spacer
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INTEGRATED DNA TECHNOLOGIES
GEN1: For General Purpose and Minimizing Primer-Dimers
• We recommend use of GEN1 rhPrimersfor most needs
• Most appropriate for standard genotyping applications and for multiplexed amplification
• Robust and works well with low levels of RNase H2 enzyme
• Mismatch guideline:M base should be the same base as the template strand
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INTEGRATED DNA TECHNOLOGIES
GEN2: For Rare Allele Genotyping
• Most appropriate for rare-allele detection or for applications where extremely high fidelity of template amplification is desired
• May require use of higher amounts of RNase H2 enzyme (range is 1–100X that needed for GEN1 rhPrimers; titration and optimization needs to be performed for each GEN2 rhPrimer set)
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Applications
Prenatal diagnostics
Cancer diagnostics• Mutations in DNA circulating in the plasma can serve as biomarkers of early tumor
development and the potential response to treatment
Pharma• Distinguishing splice variants
• Genotyping
Hospital labs• Distinguishing closely related microbial species
High throughput gene expression with pre-amplification
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INTEGRATED DNA TECHNOLOGIES
Real World Applications of GEN1 rhPrimer Designs
rhPCR
• For SNP detection
• To reduce primer-dimers in HCV assay
• For detection in a xenogenic background
• To improve multiplexing
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103 bp synthetic amplicon, run using 2 x 106 copies of templateFor AGCTCTGCCCAAAGATTACCCTG
Rev CTGAGCTTCATGCCTTTACTGT
rhPrimer-For AGCTCTGCCCAAAGATTACCCTGaCAGC-x
rhPrimer-Rev CTGAGCTTCATGCCTTTACTGTuCCCC-x
Probe: FAM-TTCTGAGGCCAACTTCCACTGCCACTTA-FQ
qPCR: rhPrimers Versus Unblocked Primers
No RNase inhibitor was present in these reactions. Even though these primers have an RNA residue, non-specific cleavage by single-stranded RNases (like RNase A) or alkaline hydrolysis will not cause background signal or false-positive amplification, because cleavage by these routes leaves a 3′-phos, which blocks primer function.
2-step PCR60oC Anneal/Extend 30 sec95oC Melt 10 sec
2.6 mU RNase H2
Anneal/cleavage/dissociation/polymerase extension can take place in the same time frame with comparable kinetics as using unmodified primers.
PCR Cq values are unchanged
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INTEGRATED DNA TECHNOLOGIES
Reduce Primer-Dimer FormationUse of rhPrimers in HCV Assay
• Typically, “intelligent” primer design can reduce the incidence of primer-dimers and false priming events. Nevertheless, these unwanted events still happen.
• Sometimes, it is necessary to design primers to specific sequences as dictated by the target sequence available.
• The following HCV assay was cited in a Roche patent (US06001611), relating to the problem of primer-dimer formation.
• rhPrimers and RNase H2 solved the problem of primer-dimer formation.
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INTEGRATED DNA TECHNOLOGIES
Unmodified primers gave the same false products with or without target.Only rhPrimers + RNase H2 correctly gave true positive products.
- - - - + + - - - - + + Rat cDNA
- - + + + + - - + + + + Target
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50
100150
250
Primersalone U R U R U RU R U R U R
Primer-dimers
Primers
True positive
Plus RNase H2Minus RNase H2
Reduce Primer-Dimer FormationHCV Subtype 1b qPCR Assay—The Solution
U = unmodified primersR = rhPrimers
L
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Detection in a Xenogenic BackgroundrhPrimers Improve Specificity in Hras qPCR Assays
• A human qPCR assay was tested for reaction specificity comparing activity in:
Human (HeLa) cDNA
vs.
Rat spinal cord cDNA
• Assays were initially run using the standard 40 cycles and then longer runs were done using 60 cycles
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INTEGRATED DNA TECHNOLOGIES
Detection in a Xenogenic BackgroundSpecificity: Human HRAS qPCR Assay in Human vs. Rat cDNA
rhPrimers
ACCTCGGCCAAGACCCgGCAG-x
||||||||| | | | ||
ACCTCGGCCTACGGCAGCTAG
CCTTCCTTCCTTCCTTGCTTCCgTCCT-x
||| | | |||||| |||||| |
CCTCCTCT..TTCCTTCCTTCCGACAG
HRAS HumanF: ACCTCGGCCAAGACCC
||||||||| | |
HRAS RatF : ACCTCGGCCTACGGCA
HRAS HumanR: CCTTCCTTCCTTCCTTGCTTCC
||| | | |||||| |||||
HRAS RatR: CCTCCTCT..TTCCTTCCTTCC
Alignment of Human Primers with Rat Hras Gene
Unmodified Primers
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INTEGRATED DNA TECHNOLOGIES
SYBR® rhPCR at a SNP in the Human SMAD7 GeneUnblocked Primers Do Not Show Any Useful Specificity
Sequence C/C T/T DCq
CAGCCTCATCCAAAAGAGGAAA 27.3 26.2
CAGCCTCATCCAAAAGAGGAAAC 26.0 28.0 2.0
CAGCCTCATCCAAAAGAGGAAAT 26.7 25.8 0.9
CAGCCTCATCCAAAAGAGGAAACA 26.1 28.2 2.1
CAGCCTCATCCAAAAGAGGAAATA 26.1 25.6 0.5
C-For + Rev T-For + Rev
C/C, C/T, and T/T DNAs
C/C
T/T and C/T DNAs
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INTEGRATED DNA TECHNOLOGIES
SYBR® rhPCR at a SNP in the Human SMAD7 GenerhPrimers Show High Specificity
For the greatest amount of discrimination, the SNP should always be placed opposite the RNA base
rs4939827 C-Block: CAGCCTCATCCAAAAGAGGAAAcAGGA-x
rs4939827 T-Block: CAGCCTCATCCAAAAGAGGAAAuAGGA-x
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INTEGRATED DNA TECHNOLOGIES
Multiplex PCRrhPrimers Improve Performance
rhPrimers (blocked primers) allow for multiplex reactions, which are otherwise impossible
Image courtesy of Jenn Jakubowski, Empirical Bioscience
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Second Generation Cleavable Primer Designs Improve Specificity for SNP Assays
First Generation design (GEN1):
CAGCCTCATCCAAAAGAGGAAAcAGGAm-x “DDDDmx” primers
Second Generation design (GEN2):
CAGCCTCATCCAAAAGAGGAAAcAxxAm “DxxDm” primers
Non-nucleotide groups make cleavage less efficient but more specific
Mismatch guideline: m base should be the same base as the template strand
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INTEGRATED DNA TECHNOLOGIES
Detection of Rare Variants in a Mixed SampleGenomic DNA samples that were homozygous for the 2 SMAD7 alleles were mixed at different ratios to determine the limit of detection of the “rare” allele in a mixed sample
Input “mismatch”-SNPInput “match”-SNP
0+666
0+66
0+6
0+0
66000+666
66000+66
66000+6
66000+0
Control(unmodified)
28.7 32.2 35.9 - 21.1 21.1 21.2 21.0
GEN1: rC-AGGAx 28.1 31.4 35.4 - 28.4 30.7 31.3 31.3
GEN2: rC-AxxA 27.9 31.4 34.6 - 28.8 31.9 34.8 37.9
GEN1: rU-AGGAx 28.1 31.3 35.7 - 27.9 30.4 31.1 31.2
GEN2: rU-AxxA 28.2 31.9 36.1 - 28.9 32.4 36.3 39.0
1:1000 1:10,0001:100
GEN2 cleavable primers are able to distinguish the presence of 1:10,000 of the “match” allele in an large background of the “mismatch” allele
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Published Papers Describing rhPCR
August 2011 BMC Biotechnology
November 2013 Journal of Biological Chemistry
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Thanks to All the Scientists Whose Work was Presented Today!
Joseph DobosyScott Rose
Mark BehlkeLing HuangSue Rupp
Kristin LongKristy Powers
Joe Walder
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INTEGRATED DNA TECHNOLOGIES
Additional Questions?
Email: [email protected]
For more information or to order rhPrimers and the RNase H2 enzyme: www.idtdna.com/RNaseH2.
To view articles on rhPCR or request a copy of DECODED 4.2—a special qPCR edition of the IDT newsletter:www.idtdna.com/DECODED