Challenges of PCR-based genotyping
Identifying and characterizing single nucleotide polymorphisms (SNPs) is important for many applications, including the study of gene function, identification of specific species or individuals, diagnosis of disease caused by gene variation, and targeted medical intervention based on specific genotypes. 5′-nuclease PCR assays are a fundamental technology for genotyping, because they allow labs to inexpensively, yet accurately, determine genotype using high-throughput processing and low sample input.
Another advantage of PCR methods is that they provide highly robust amplification of specific target sequences. However, non-specific amplification can also occur when reaction conditions are not optimal or when primers recognize sequences that are similar to the desired target.
For some PCR applications, obtaining a majority of correct amplification product is sufficient. However, for 5′-nuclease genotyping applications, non-specific amplification and primer-dimers can reduce peak signal levels for assays, leading to difficult data interpretation or even unusable data.
Reduction of off-target amplification with rhPCR
To increase target specificity in PCR-based applications, IDT scientists developed RNase H2–dependent PCR (rhPCR). rhPCR relies on an RNase H2 enzyme from Pyrococcus abyssi that removes RNA bases from a DNA duplex, only when the RNA bases are paired to their correct DNA complement. Importantly, cleavage by RNase H2 leaves an intact, 3′ hydroxyl group that can be used for extension by a DNA polymerase (Figure 1).
The P. abyssi RNase H2 functions optimally in conditions that are compatible with thermophilic DNA polymerases, including Taq polymerase. This led the founder of IDT, Dr Joseph Walder, and a team of researchers to develop a novel method for reducing off-target amplification in PCR. The research included development of rhPCR primers that contain a single RNA base close to the 3′ end of each primer and a 3′ modification that blocks extension. During a normal PCR cycle, the RNase H2 enzyme removes the RNA base and blocking group from the primer end, only when the primer has bound the correct target, thus allowing for extension (Figure 2). IDT researchers have also extensively tested which position is optimal for the placement of the RNA base .
The rhAmp Genotyping System
The rhAmp SNP Genotyping System uses the rhPCR primer technology as described above for SNP-specific assay primers and a locus-specific reverse primer. To further enhance the method, rhAmp SNP Genotyping includes several other technologies to create a highly precise genotyping solution. The system adds an optimized reporter mix containing universal probes for the reference and alternate alleles in the 5′-prime nuclease PCR assays. The forward primers in the rhAmp SNP Genotyping Assay also add a tail sequence to each amplicon that is recognized by the universal reporter system. Importantly, the probes recognize the complement of the primer tail, which is created during amplification. This means that the probes cannot interact with the assay primers directly (Figure 4).
Advantages to using a universal reporter
The first advantage is cost. The most expensive component of other genotyping assays is the gene-specific, fluorescently-labeled probe. Another advantage is that the rhAmp Genotyping System assays can be designed with amplicons as short as 40 bp, because the probe does not need to bind between the 2 assay primers. This creates flexibility that is not available with larger amplicons in challenging genomic regions. Finally, the 2 universal reporter probes are highly optimized for universal cycling conditions. This means you don't have to worry about optimizing different conditions for each assay, thus eliminating a significant issue of high-throughput applications.
Genotyping-optimized Taq polymerase
The rhAmp Genotyping System also requires the use of the rhAmp Genotyping Master Mix. In addition to the RNase H2 enzyme previously described, this mix contains a novel Taq DNA polymerase that outperforms other polymerases in SNP genotyping assays (Figure 5). Wild-type Taq polymerase is sensitive to mismatches at the 3′ base of PCR primers, but it will still amplify from some incorrectly annealed primers. Even a very small number of incorrect priming extensions by Taq creates new erroneous template that can be further amplified. If enough wrong template is created and amplified, the competing degradation of the probe for an allele that is not present in the sample can cause significant convolution of correct and incorrect fluorescent signals. This is visible as a decrease in the cluster angle-of-separation for the genotypes in the resulting allelic discrimination plots (Figure 5B). The mutant Taq polymerase in the rhAmp Genotyping Master Mix is much more sensitive to allelic mismatches at the 3′ base of the primer, resulting in improved genotyping performance (Figure 5A).
Complete rhAmp SNP Genotyping System
The rhAmp SNP Genotyping chemistry is only compatible with rhAmp SNP Assays. The current rhAmp SNP Assay database for human SNPs is very large, with >10 million designs available. There is also a selection of assays that have been further validated in the lab for SNPs involved in the absorption, distribution, secretion, and excretion (ADME) of pharmaceutical compounds. If you are working in other species, including plant species, you can easily design assays using the custom rhAmp Genotyping Design Tool.
You can also easily design gBlocks® Gene Fragment controls during the ordering process. For SNPs in challenging sequence regions, using such controls can help data analysis software separate genotypes and, in some cases, prevent loss of data.