Amplification and reporter mixes optimized for rhAmp SNP GenotypingrhAmp 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¡Ç nuclease reporter system to distinguish the reference allele from the alternative allele for any rhAmp SNP Assay.
rhAmp¢â Genotyping Master MixA 2X master mix solution, containing the required enzymes and additional components necessary for activation and amplification of rhAmp SNP Assays.
rhAmp¢â Reporter MixA 40X universal, reporter probe mix for use with rhAmp¢â Genotyping Master Mix and rhAmp SNP Assays. Does not contain reference dye.
rhAmp¢â Reporter Mix w/Reference A 40X universal, reporter probe mix with reference dye required by certain instruments. For use with rhAmp¢â Genotyping Master Mix and rhAmp SNP Assays.
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¡Ç 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¡Ç 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¡Ç-hydroxyl group for extension by Hawkeye¢â Taq Polymerase. RNase H2 cleaves the phosphate backbone between the DNA base and RNA base during normal PCR cycling conditions. This produces a 3¡Ç-hydroxyl group on the DNA base that is accessible to DNA polymerase extension.
rhAmp Genotyping Master MixrhAmp Genotyping Master Mix contains hot start versions of RNase H2 from Pyrococcus abyssi (P.a.), and a novel Hawkeye¢â 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, P.a. RNase H2 enzyme has optimal activity between 70¡ÆC and 75¡ÆC and is functional in rhPCR between 50¡ÆC and 75¡ÆC. 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 Hawkeye Taq DNA Polymerase is a novel, proprietary polymerase that is optimized for allelic discrimination. The Hawkeye 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 MixrhAmp Reporter Mix is a cost-effective, universal, 5¡Ç 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¢ç universal probe is assigned to the alternate allele. The Yakima Yellow reporter dye can be read on qPCR instruments in the VIC¢ç 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¡Ç nuclease SNP assays, the rhAmp SNP universal reporter system eliminates the expense of synthesizing two SNP-specific probes for every assay. This system enables rhAmp SNP Assays to provide higher quality genotypes at a more affordable price.
Table 1. rhAmp¢â Reporter Mix, with or without reference dye, matched to correct size rhAmp Genotyping Master Mix.
* For use with real-time qPCR instruments that do not require reference dye. Table 2. Reference dye requirements for various PCR systems. For instruments not listed, please check with the manufacturer.
protocol_rhAmp_SNP_Genotyping.pdf (1,063,651kb) |
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