Oligoribonucleotide interference-PCR: principles and applications

Abstract

PCR amplification of multiple templates using common primers is used widely for molecular biological research and clinical diagnosis. However, amplifying a specific DNA sequence harboring a mutation that is present in a small number of mutant cells within a large population of normal cells (e.g., as in cancer) in a tissue is difficult using the original PCR protocol. Thus, some measures are necessary to suppress amplification of background signals. To achieve this, we developed the oligoribonucleotide (ORN) interference-PCR (ORNi-PCR) technology in which an ORN (short RNA) hybridizes with a complementary DNA sequence to inhibit PCR amplification across the specific target sequence. ORNs can be prepared inexpensively, and ORNi-PCR can be carried out easily by adding ORNs to the PCR reaction mixture. Suppressing amplification of target sequences by ORNi-PCR is useful for detecting target sequence mutations. We showed that ORNi-PCR can discriminate single nucleotide mutations in cancer cells and indel mutations introduced by genome editing. We also showed that ORNi-PCR can identify the CpG methylation status of a target sequence within bisulfite-treated DNA, and can enrich DNA sequences of interest from a DNA mixture by suppressing amplification of unwanted sequences. Thus, ORNi-PCR has many potential applications in various fields, including medical diagnosis and molecular biology. In this review, we outline the principles of the ORNi-PCR method and its use to detect nucleotide mutations in a variety of specimens.

PCR Techniques and Their Clinical Applications

“…Examples include multiplex PCR, broad-range PCR, strip PCR, and direct strip PCR methods, which are used to detect 24 pathogens that cause ocular infections [45]. Oligoribonucleotide interference PCR (ORNi-PCR) can amplify the desired transcript variant in cDNA by inhibiting the amplification of unwanted transcripts and can simultaneously detect two single-nucleotide mutations in the same allele of epidermal growth factor receptor (EGFR) gene in lung cancer cells with high sensitivity, which is expected to be used for the early detection of cancers caused by single-nucleotide mutations [46]. Furthermore, there are other types of PCR, such as methylation-specific PCR, type-specific PCR, etc.…”