Oligoribonucleotide interference-PCR: principles and applications.

Abstract

Polymerase chain reaction (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.