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).
Figure 1. RNase H2 cleavage produces a 3′-hydroxyl group for extension by a novel Taq DNA polymerase that is optimized for SNP genotyping. RNase H2 cleaves the phosphate backbone between DNA and RNA bases under normal PCR cycling conditions. The cleavage reaction leaves a 3′-hydroxyl group on the DNA base that is accessible for DNA polymerase extension.
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 .
Figure 2. Novel rhPCR method employs RNase H2 to increase amplification specificity. Use of RNase H2 to incorporate a specificity check into the PCR primers dramatically reduces non-specific amplification and primer-dimer artifacts, even in highly multiplexed reactions (Figure 3). This method forms the core of the rhAmp SNP Genotyping System, and the resulting impressive performance has implications for many PCR-based applications.
Figure 3. rhPCR primers virtually eliminate primer-dimers and nonspecific amplification artifacts in multiplex PCR. In multiplex PCR amplification of 96 targets from human genomic DNA (NA12878, Coriell Institute), a set of multiplex primers for the 96 assays (192 individual primers) was synthesized as standard PCR primers and as RNase H2-activated, rhAmp primer pairs. The results of the assays that used rhAmp primer pairs show tight control of primer-dimer artifacts, even in these highly multiplexed assays compared with the standard PCR primer reactions. Total reaction volumes (50 µL) included 10 ng genomic DNA template or no template (no template controls, NTC). RNase H2 was present in all reactions, but had no functional role in PCRs with standard primers.
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).
Figure 4. rhAmp SNP Genotyping PCR. All components needed to measure both alleles are combined in a single reaction before cycling. (A) Both allele-specific primers query the SNP locus. (B) RNase H2 enzyme cleaves the primers that are perfectly annealed to the target sequence, removing the RNA base and 3’ blocking modification, which allows extension by the Taq Polymerase. (A–C) During the first two amplification cycles, a tail sequence is incorporated into the amplicon that continues to be included in amplicon generation for the remainder of programmed cycles. (D) Tail sequences are subsequently recognized by a probe-based, universal reporter system (Universal probe 2 not shown). (E) Polymerase extension leads to degradation of the probes and signal generation.
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).
Figure 5. A novel genotyping Taq DNA polymerase provides superior allelic discrimination. rhAmp Genotyping Master Mixes, formulated with (A) a novel Taq DNA polymerase developed by IDT or (B) wild type Taq DNA polymerase were compared using a rhAmp SNP assay (rs2269829), targeting an intronic SNP in PON1 and 3 ng of gDNA samples from 46 individuals (Coriell Institute). Analysis was performed using the QuantStudio™ 7 Flex Real-Time PCR System software (Thermo Fisher). With improved selectivity at the SNP site, the rhAmp Genotyping Master Mix containing the IDT Taq DNA polymerase results in lower off-target signal and greater cluster angle separation.
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.