rnase h2 pcr—a new technology to reduce primer dimers and increase genotyping accuracy

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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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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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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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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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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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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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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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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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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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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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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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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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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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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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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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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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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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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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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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

mailto:[email protected]

http://www.idtdna.com/RNaseH2

http://www.idtdna.com/DECODED

rnase h2 pcr—a new technology to reduce primer dimers and increase genotyping accuracy

Science

target dna sequence

identical dna blue

multiplexed amplification

amplification targets

ensuing pcr

generation of rhprimers

rare alleleequal amplification

abyssi rnase h2 activity0