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.
rhAmp Genotyping Master Mix
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
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 sizes are configured to complement rhAmp Genotyping Master Mix product sizes for easy planning and ordering (Table 1)
- Available with or without reference dye for maximum flexibility on all qPCR platforms (Table 2)
Table 1. rhAmp Reporter Mix, with or without reference dye, matched to correct size rhAmp Genotyping Master Mix.
rhAmp Reporter Mix*
or
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
|
Table 2. Reference dye requirements for various PCR systems.*
| | Reference dye required?
|
| PCR system
| Yes
| No
|
| 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
|