RNase H2–dependent PCR
While it is possible to achieve high specificity with PCR amplification, project objectives sometimes require primer positioning at suboptimal locations within the target. This can result in the formation of primer-dimers and/or undesired amplification of homologous sequences.
To increase PCR specificity and eliminate primer-dimers, scientists at IDT developed RNase H2–dependent PCR . The technology relies on cleavage of a 3′-blocked primer by Pyrococcus abyssi (P.a.) RNase H2 to activate the primer before target amplification. RNase H2 cleavage occurs at the 5′ end of a single RNA base included in the blocked primer, and only after primer hybridization to target DNA (Figure 1).
Because primers must hybridize to the target sequence to form RNA:DNA hybrids before cleavage and primer activation can occur, the primers are unable to form primer-dimers. Additionally, the requirement for high target complementarity reduces amplification of closely related sequences. After cleavage, the unblocked primer is available to support extension and replication during PCR. RNase H–dependent PCR, historically referred to as rhPCR, forms the basis of rhAmp technology, which facilitates SNP genotyping applications, highly multiplexed amplification, and the detection of rare alleles.
Figure 1. RNase H-dependent PCR.
Application of RNase H–dependent PCR: Detecting alternatively spliced latrophilin transcripts
The recent paper, Latrophilins function as heterophilic cell-adhesion molecules by binding to teneurins: Regulation by alternative splicing, from Boucard AA, Maxeiner S, and Südhof TC , provides an example of using RNase H–dependent PCR for measuring the expression of transcripts that undergo complex splicing. It also extends the method to RT-PCR. In mouse, the latrophilin genes (Lphn1, Lphn2, Lphn3; see Definitions sidebar) each include a small, alternatively spliced mini exon of only 12–15 bp (Figure 2). This exon is highly conserved across species in invertebrates and vertebrates, suggesting that it encodes a region of functional significance.
Figure 2. qPCR assay strategy for detecting alternative splicing of latrophilin. The 3 possible splicing patterns of the latrophilin genes are shown (top) with the resulting transcripts and qPCR assay strategies (primer and probe locations) for their detection (3 transcripts at bottom).
The similarity between latrophilin genes makes it difficult to use standard qRT-PCR assay design tools to generate assays that would distinguish the various transcripts. To complicate matters further, the scientists discovered that for Lphn2, the mini exon had 2 potential alternative splice acceptor sites, leading to shortened (12 bases, 4 amino acids) and full (15 bases, 5 amino acids) mini exon variants (Figures 2 and 3). This proved to be a situation where the RNase H–dependent PCR could provide an advantage.
Boucard et al. designed blocked, cleavable forward primers with the sequences at their 3′ ends positioned:
- just within the start of the full-sized mini exon
- just within the start of the shortened mini exon
- across exon boundaries excluding the mini exon
Figure 3 shows these primer sequence locations relative to the latrophilin exon boundaries. The scientists then modified the original RNase H–dependent PCR technique  for RT-PCR, by adding an optimized concentration of RNase H2 after the initial cDNA synthesis step . They were able to design these specific assays within a limited design region to distinguish between alternatively spliced variant transcripts, allowing them to compare Lphn gene expression levels in various tissues.
Figure 3. Design of primer and probe sequences for RNase H–dependent PCR. Shown are the respective parts of the transcript sequences for latrophilins (Lphn1, Lphn2, and Lphn3) with the forward primers (labeled a, b, c) used in the RNase H–dependent PCR assays depicted above and below. Distinct blocked, cleavable forward primers were designed to specifically amplify transcripts with or without the alternatively spliced mini exon. Sequence displayed in capital letters represents DNA bases, while lowercase letters are the RNA bases where RNase H cleavage occurs. The “x” at the 3′ end of each transcript represents the C-3 spacer used as the primer blocker. Dual-Quenched Probes, which are the standard probe type in PrimeTime qPCR Assays are shown in green; green open circles = dyes, red circles = quenchers. Red bases = sequence common to transcripts of all 3 forms of latrophilin; blue bases = sequence common to 2 forms of latrophilin; black bases = sequence unique to that specific latrophilin transcript.
A role for the alternatively spliced mini exon?
The resulting quantitative RT-PCR assays selectively measured Lphn1 and Lphn3 mRNAs containing or lacking the mini exon insert, and Lphn2 mRNAs containing either the shorter or longer version of the mini exon insert or lacking the insert all together (Figure 3). Expression results of latrophilin variants revealed that alternative splicing of the mini exon differs among latrophilins and that, in particular, most Lphn2 mRNAs in brain contain the insert, whereas most Lphn2 mRNAs in non-neural tissues lack it.
Saturation binding experiments performed on Lphn1 splice variants revealed that the sequence encoded by the mini exon had a differential effect on Lphn1 binding affinity to its endogenous ligands. Indeed, insertion of the mini exon affected Lphn1 binding affinity to teneurins, but not to Flrt3 or neurexin1 (see the Definitions sidebar) . This activity suggests that the effect of alternative splicing on ligand binding is specific and that this region appears to further modulate protein-protein interaction.
rhAmp PCR: A novel approach to improve genotyping, multiplexing, and NGS library preparation
rhAmp PCR technology is ideal for distinguishing highly similar sequences, including in genotyping (see rhAmp SNP Genotyping System). In addition, rhAmp PCR technology can increase the success of methods currently hindered by unwanted interactions between multiple primer sets (e.g., primer-dimers), such as in highly multiplexed qPCR assays and in library construction for next generation sequencing, where resulting libraries are often contaminated with primer-dimer “blank reads”. The technique is also useful for rare allele detection, where the added specificity provided by the blocked-cleavable primers enables detection of a rare mutant allele in a background of large amounts of wild-type DNA.
RNase H2 can be incorporated into current end-point PCR and qPCR protocols with little to no modification in reaction temperatures, cycling times, or analysis procedures. Primer design instructions, a protocol for RNase H–dependent PCR, and published examples of applications using this technology can be found on the rhPCR primers and RNase H enzyme web pages.