Genomics research and discovery has led to a large increase of reported single nucleotide polymorphisms (SNPs). From 2006 to 2017, the number of refSNPs in the NCBI dbSNP database has increased 13-fold. Many polymorphisms can be linked to disease susceptibility and responses to chemical therapies. Other polymorphisms are used as trait identifiers in livestock and plants. Being able to inexpensively and accurately determine the genotype in high-throughput fashion, with low sample input is a critical need in current, large-scale screening efforts. In this presentation, we present a novel, probe-based, PCR genotyping solution that possesses the universal cycling conditions, strong signal generation, and benchtop reaction stability needed for high-throughput screening. We also present the mechanism and unique technical advantages of using the rhAmp SNP Genotyping System, and we will illustrate how easy it is to generate high quality genotyping data.

1. rhAmp™ SNP Genotyping: A novel approach
for improving PCR-based SNP genotyping
Scott Rose, PhD
1

2. Agenda
• Principal, rhPCR technology introduction
• rhAmp™ SNP Genotyping: what it is and how does it work?
• Technical highlights
• Design tool description
• Product description and features
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3. RNase H2 is at the core of the foundational rhPCR technology
• The RNase H2 enzyme from the hyperthermophilic archaeon
Pyrococcus abyssi:
– Is an endoribonuclease that binds to RNA-DNA duplexes
– Will cleave a single ribonucleotide residue embedded within a
heteroduplex
– Cleaves the RNA containing strand leaving a 5′-phosphate and a 3′-
hydroxyl group
– Will not cleave single-stranded RNA
– Functions optimally at high temperature, with monovalent and divalent
cation requirements that make it ideal for use with DNA polymerases in a
single-tube format
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4. RNase H2 cleavage mechanism
1. Recognizes and binds to
heteroduplex
2. Cleaves at DNA-RNA junction of
the RNA containing strand
3. Leaves the terminal DNA base with
a 3′ hydroxyl (OH)
4. RNA:DNA duplexes with
mismatches opposite to the RNA
base as well as nearest neighbor
bases are poor substrates
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5. Unique DNA:RNA hybrid primers require a perfect match
for efficient primer activation by RNase H2
5
rN

Blocked primers annealed to
matched target are cleaved by
RNase H2
Forward rhPCR primer
Reverse rhPCR primer
Forward PCR primer
Reverse PCR primer
rN
PCR Product
Amplification by any DNA
polymerase
3′ blocker
rN RNA base

6. rhPCR technology greatly reduces primer-dimers
and spurious amplification due to mispriming
• Improves signal to noise
• Increases multiplexing
capabilities
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• rhPCR primers in a 96-plex PCR reaction
• PCR was performed using 10 ng of
purified human gDNA (Coriell Institute)

7. rhPCR-based, rhAmp SNP Genotyping System
• The rhAmp SNP Genotyping System provides a simple, accurate, and cost-
effective technology for fluorescence-based genotyping in real time or end-point
modes.
• Enhanced allele discrimination is achieved with a powerful 2-enzyme mismatch
recognition system.
– RNase H2 enzyme provides a sequence specific cleavage event
– Superior allelic discrimination obtained with IDT’s novel mutant Taq DNA Polymerase
• Cleavable, blocked, assay primers are generated through an advanced primer
design pipeline.
– Fluorescent signal generation by universal reporter probes
• rhAmp SNP Genotyping uses a standard 3-step thermal cycling profile, is
platform agnostic, and compatible with automation
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8. rhAmp SNP Genotyping schematic
• Step 1: Primer design and annealing
– Modified primers include a 3′-blocking group and an RNA base next to the SNP site.
– Primers must be annealed to the matched target for RNase H2 cleavage to occur, resulting in deblocking of primers.
• Step 2: Primer cleavage and extension
– Primer cleavage by RNase H2 generates a 3′-OH group that is extendable by the highly discriminating, mutant Taq polymerase.
• Step 3: Universal reporter incorporation
– rhAmp primers have 5′ tails, which incorporate a universal 5′-nuclease assay reporter system.
• Step 4: Probe cleavage and signal generation
– The FAM and Yakima Yellow® (Elitech Group) reporter dyes distinguish the 2 alleles. 8

9. Novel, IDT mutant Taq DNA Polymerase provides improved, allele-
specific PCR capabilities in conjunction with RNase H2
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Optimized, rhAmp primer design
IDT mutant Taq polymerase
Optimized, rhAmp primer design
wild-type Taq polymerase

10. Higher signal and improved cluster separation
• Robust, consistent
signal improves
cluster angles and
separation
• Greater cluster
separation provides
higher confidence for
automated and
manual calls
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CYP1A2 (rs762551) CYP3A5 (rs776746)
Allele A Allele AAlleleC
AlleleG
rhAmp SNP Assay
5′-nuclease genotyping assay
FAM or Yakima Yellow® dyes, 3 ng gDNA (Coriell Institute)

11. ADME gene CYP3A5 (rs776746)
rhAmp SNP Assay 5′-nuclease genotyping assay
Higher, more uniform, raw fluorescent signals
• Optimized universal
probe design enables
maximum probe
cleavage and signal
generation
• 3 ng of human gDNA
from 5 individuals with
heterozygous genotype
(Coriell Institute)
• Yakima Yellow dye is
read in the VIC (Thermo
Fisher) dye channel—no
recalibration needed
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12. Consistently high performance across instrument platforms
• 3 ng of purified human gDNA from 46 individuals (Coriell Institute)
• Reaction volumes range from nanoliters to microliters
• Yakima Yellow signal was detected using the VIC channel without recalibration
12
QuantStudio™ 7 CFX Biomark™

13. rhAmp SNP Genotyping with gDNA isolated from blood
rhAmp SNP Assays provide consistent performance across sample types. Assays for were designed for
SNPs in genes (A) SLCO1B1 and (B) ABCC2. These assays were used to assess SNPS in 3 different sample
types: synthetic gBlocks® Gene Fragments (*), purified genomic DNA (Coriell Institute; ∆) and genomic DNA
isolated from whole blood using the Qiagen QIAamp DNA Blood Mini Kit (prepared by BioChain Institute; ○).
Assay performance is similar across sample types, demonstrated by overlapping symbols. Analysis was
performed using QuantStudio™ 7 Flex Real-Time PCR System software (Thermo Fisher). 13

14. rhAmp SNP Genotyping with crude maize lysate in low-
volume PCR
• Maize SNP
marker
• 96 crude, maize
lysate, DNA
samples
• <2 μL PCR
reaction volume
• Note the
difference in
signal intensity
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Tighter clusters and higher signals with rhAmp SNP Assays
AlleleG
7
6
5
4
3
2
1
AlleleG
1.0 1.5 2.0 2.5 3.0 3.5
Allele C
0.20
0.15
0.10
0.05
0.025 0.050 0.075 0.100 0.125
Allele C
Other 5′ nuclease assayrhAmp™ SNP Genotyping

15. CYP2C9 (rs72558187)
10 ng of human gDNA purified
from whole blood
Analysis with 1000 copies of
gBlocks® controls for each cluster
gBlocks Gene Fragments are available as positive control
sequences
• Manual and automatic
calls are improved with
control samples for each
cluster
• gBlocks Gene Fragments
controls can be designed
specifically for each
assay
• www.idtdna.com/gBlocks
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16. rhAmp Genotyping high performance design tool features
• 10 million predesigned assays for human SNPs
• Custom design tool for AgBio and other species
• Assays delivered within 7 business days
1
6
1. Select your assay type
2. Select your species
3. Select your target
input format
4. Copy and paste your
target list

17. Design assays for any species using FASTA sequence entry
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18. Complete rhAmp SNP Genotyping System
• >10 million predesigned assays for human SNPs
• High performance, custom assay design tool for other species
• Experimentally validated SNP assays targeting ADME genes
• Dual-enzyme master mix, optimized for superior discrimination
• Universal reporter system with optional reference dye
• Optional, synthetic gBlocks Gene Fragment controls
• Simple, one-tube chemistry works on all common qPCR platforms
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19. rhAmp SNP Genotyping System details
• Order in tubes or 96-well matrix racks (minimum of 24 assays per rack)
• Choose from 4 reaction scales for assays: 100, 750, 2500, or 6000
reactions
• Multiple configurations of Master Mix and Reporter Mix to match your
instrument and the size of your experiment
• Validate markers affordably using the smallest unit size commercially
available (100 reactions) and value pricing across other unit sizes
• Core primer sequences provided with each order
• gBlocks Gene Fragments sequences available as control templates
• Assays shipped within 7 business days
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20. THANK YOU
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