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 (rhPCR) . The technology relies on cleavage of a 3′-blocked primer by RNase H2 from Pyrococcus abyssi (P.a.) 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 for cleavage and primer activation to occur, they are unable to form primer dimers and the requirement for high target complementarity reduces amplification of closely related sequences. Once cleaved, the unblocked primer is available to support extension and replication during PCR. The rhPCR amplification technology facilitates SNP genotyping applications, high levels of multiplexed amplification, and the detection of rare alleles.
Figure 1. RNase H-Dependent PCR (rhPCR).
rhPCR application: Detecting differential amplification of 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 rhPCR use for measuring the expression of transcripts that undergo a complex splicing pattern. It also extends the rhPCR 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 above with the resulting transcripts and qPCR assay strategies (primer and probe locations) for their detection, below.
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 & 3). This proved to be a situation where the rhPCR technique could provide an advantage.
Boucard et al. designed blocked, cleavable forward primers such that the sequences at their 3′ ends were 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 details these primer sequence locations relative to the latrophilin exon boundaries. The scientists then modified the original rhPCR technique  for RT-PCR, by addition of 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 the Lphn gene expression levels in various tissues.
Figure 3. Design of RNase H-dependent RT-PCR primer and probe sequences. Shown are the respective parts of the transcript sequences for latrophilins (Lphn) 1, 2, and 3 with the forward primers (labeled a,b,c) used in the RNase H-dependent RT-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 those letters not capitalized are the RNA bases where RNase H will cleave. The “x” symbol at the 3′ end of each transcript represents the C-3 spacer used as the primer blocker. The Dual-Quenched Probes that come standard with IDT 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.
rhPCR: A novel approach to improve genotyping, multiplexing, and NGS library preparation
In addition to its use in distinguishing highly similar sequences, including in genotyping, the assay should increase the success of methods currently hindered by unwanted interactions between multiple primer sets (e.g., primer-dimers, etc.), such as in highly multiplexed qPCR assays and library construction for Next Generation DNA Sequencing, where resulting libraries are often contaminated with primer-dimer “blank reads”. The method 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 in current end-point PCR and qPCR methods with little to no modification in reaction temperatures, cycling times, or analysis procedures. IDT provides primer design instructions, including specific details for SNP detection. These recommendations, along with an rhPCR protocol and additional published examples of applications using this technology, can be found on the rhPCR primers and RNase H enzyme webpages.
Synaptic cell adhesion molecules that are type I transmembrane proteins, comprised of 3 genes in vertebrates. Each of these genes is controlled by 2 different promoters, resulting in a- and b-neurexins. A hallmark of neurexin genes is their extensive alternative splicing, which can generate more than 3000 different isoforms; and can modulate protein-protein interactions [12–14] and, by consequence, neuronal functions .
A fibronectin leucine-rich transmembrane protein involved in complex protein-protein interaction networks. Genetic ablation of FLRT3 causes defects in tissue morphogenesis and development as well as synaptic defects [7,15]. Although FLRT3 is linked to neuronal functions, its precise role is unclear, as it appears to act both as a repulsive guidance cue as well as being synapse inducing .
Teneurin (or Odz)
A family of 4 large type II transmembrane proteins (in vertebrates) that owe their name to a similarity with the tenascin proteins. Mouse gene deletion and overexpression studies suggest these proteins play a role in axonal guidance, oligodendrocyte differentiation, myelination, and synapse target selection [5,6,9–11].
Latrophilins (or calcium-independent receptors for α-latrotoxin (CIRLs))
G protein-coupled transmembrane receptors (GPCRs) that exist as 3 isoforms in mammals and are encoded by genes Lphn1, Lphn2, and Lphn3. Each gene expresses alternatively spliced variants in the N-terminal lectin binding domain  (Figure 2). While deletion of Lphn1 alone shows no major phenotype , latrophilins do appear to form functional protein complexes that affect synapse development and maintenance through binding to teneurins and FLRT3 [4–7], but also to neurexins, the calcium-dependent receptor for α-latrotoxin .