Amplification and reporter mixes optimized for rhAmp SNP Genotyping
rhAmp Genotyping Master Mix and rhAmp Reporter Mixes are specially formulated for use with rhAmp SNP Assays to deliver superior specificity and signal-to-noise ratios for high confidence genotype calls. rhAmp Genotyping Master Mix is a unique, two-enzyme formulation optimized for highly specific amplification and accurate allele discrimination. rhAmp Reporter Mixes contain a universal 5′ nuclease reporter system to distinguish the reference allele from the alternative allele for any rhAmp SNP Assay.
The rhAmp SNP Genotyping System is a complete solution requiring the following 3 components:
rhAmp SNP Genotyping technology incorporates a novel method using blocked PCR primers that contain a single RNA base. The rhAmp primers are activated when the blocking motif is removed by the RNase H2 in the rhAmp Genotyping Master Mix. Cleavage occurs at the 5′ end of the RNA base (Figure 1) only when it is perfectly matched to its DNA complement. Primer cleavage occurs in the background during each anneal/extend cycle to generate a 3′ hydroxyl for polymerase extension, which means the PCR protocol does not require any additional steps. The mechanism creates extremely tight control of amplification that greatly reduces primer-dimers and off-target amplification of closely related sequences.
Figure 1. RNase H2 cleavage produces a 3′-hydroxyl group for extension by a proprietary Taq polymerase. RNase H2 cleaves the phosphate backbone between the DNA base and RNA base during normal PCR cycling conditions. This produces a 3′-hydroxyl group on the DNA base that is accessible to DNA polymerase extension.
rhAmp Genotyping Master Mix contains hot start versions of RNase H2 from Pyrococcus abyssi (P.a.), and a novel Taq DNA Polymerase with enhanced sensitivity for allelic mismatches. This dual enzyme formulation, combined with rhAmp SNP Assays, provides highly sensitive, allele-specific PCR for the detection of single-nucleotide polymorphisms.
Pyrococcus abyssi is an extreme thermophile. Therefore, the P.a. RNase H2 enzyme has optimal activity between 70°C and 75°C and is functional in rhPCR between 50°C and 75°C. P.a. RNase H2 has very low activity at room temperature (~1000X less active). These properties allow the enzyme to deliver excellent performance in the optimized rhAmp Genotyping PCR buffer conditions.
The genotyping Taq DNA Polymerase is a novel, proprietary polymerase that is optimized for allelic discrimination. The Taq polymerase and RNase H2 combination delivers clear discrimination and high signal-to-noise ratios, due in part to reagents not being unnecessarily consumed by primer-dimers and spurious amplification products.
rhAmp Reporter Mix is a cost-effective, universal, 5′ nuclease assay reporter mix that contains 2 universal probes to distinguish the reference allele from the alternate allele and a universal forward primer. The FAM universal probe is assigned to the reference allele, and the Yakima Yellow® universal probe is assigned to the alternate allele. The Yakima Yellow reporter dye can be read on qPCR instruments in the VIC® channel without need for calibration.
The universal primer and probe designs have been optimized to deliver consistent, robust signal-to-noise for improved automated allele calling. And, unlike traditional 5′ nuclease SNP assays, the rhAmp SNP universal reporter system eliminates the expense of synthesizing 2 SNP-specific probes for every assay. This system enables rhAmp SNP Assays to provide higher quality genotypes at a more affordable price.
|rhAmp Reporter Mix*|
rhAmp Reporter Mix w/Reference dye†
|rhAmp Genotyping Master Mix matching size||Reactions‡|
|25 μL||0.5 mL (1 X 0.5 mL)||100|
|250 μL||5.0 mL (1 X 5 mL)||1000|
|500 μL||10 mL (2 X 5 mL)||2000|
|1250 μL||25 mL (5 X 5 mL)||5000|
|2500 μL||50 mL (1 X 50 mL)||10,000|
|Reference dye required?|
|7900HT Fast and 7300 Real-Time PCR System (Thermo Fisher Scientific)||X|
|StepOne™ and StepOnePlus™ Real-Time PCR System (Thermo Fisher Scientific)||X|
|Mx3005P™ and Mx4000P™ qPCR System (Agilent)||X|
|7500 Real-Time PCR System (Thermo Fisher Scientific)||X|
|ViiA™7 Real-Time PCR System (Thermo Fisher Scientific)||X|
|QuantStudio™ Flex Systems (Thermo Fisher Scientific)||X|
|CFX, iQ™, and Opticon™ Real-Time PCR Detection Systems (Bio-Rad)||X|
|LightCycler® Real-Time PCR Systems (Roche)||X|
* For instruments not listed, please check with the manufacturer.
Whether you are just starting with a genotyping study, repeating experiments, or continuing a long-term investigation, you can rely on any lot of the rhAmp Genotyping Master Mix to deliver the same, accurate, genotyping results (Figure 1).
Figure 1. Consistent genotyping results across multiple lots of rhAmp Genotyping Master Mix. 16 rhAmp SNP Assays were used in 5 µL genotyping reactions, with 3 ng purified genomic DNA samples from 46 individuals (Coriell Institute) and 3 unique lots of rhAmp Genotyping Master Mix. Genotyping results were analyzed using QuantStudio™ 7 Flex Real-Time PCR System software (Thermo Fisher). The rhAmp Genotyping Master Mix provides reproducible results that have been tested to demonstrate comparable cluster angles and cluster density with rhAmp SNP Assays.
rhAmp SNP Genotyping reactions are stable for up to 24 hr at room temperature (20–25°C) with all reagents combined before PCR cycling (Figure 2), which facilitates automated and high-throughput reaction setup.
Figure 2. rhAmp SNP Assays and Master Mix are stable after mixing for up to 24 hr at room temperature. Two identical genotyping reaction plates were prepared, using 16 rhAmp SNP Assays in 5 µL genotyping reactions, with 3 ng of purified genomic DNA samples from 46 individuals (Coriell Institute). One plate was run immediately, at time 0 hr, and the second reaction plate remained on the benchtop for 24 hr before the PCR was performed. Genotyping results were analyzed using QuantStudio™ 7 Flex Real-Time PCR System software (Thermo Fisher). The rhAmp Master Mix provides stable results up to 24 hours after reaction preparation, with no significant change in genotyping results.
rhAmp SNP Assays are compatible with all common real-time qPCR platforms (Figure 3). Universal probes are part of the rhAmp Reporter Mix, available with or without reference dye to ensure compatibility.
Figure 3. rhAmp SNP Genotyping assays deliver high precision and performance regardless of qPCR instrument used. Allelic discrimination plots for SNPs located in genes (A and B) SMARCD1 (rs3782323) and (C and D) LINC00111 (rs3850713) show consistent performance of rhAmp SNP Assays analyzed using QuantStudio Real-Time PCR Software (Thermo Fisher) or the CFX Real-Time PCR Software (Bio-Rad). Genotyping was performed in 5 µL reactions, using 3 ng human gDNA from 46 individuals (Coriell Institute). Yakima Yellow® signal was detected using the VIC® channel without calibration. More than 90% of rhAmp SNP Assays demonstrate a call rate >95% and call accuracy of 99.5% regardless of qPCR instrument.