Cleavage of double-stranded copy RNA by RNase 1 and RNase T 1 provides a robust means to detect p53 gene mutations in clinical specimens

Detecting somatic mutations in patient specimens is challenging because of the wide variation in quality and quantity of genomic DNA in clinically der ived material. in cancer specimens, the challenge of detecting mutations is usually compounded by the presence of large numbers of nonmutated normal c ells that dampen the relative signal that can be obtained from employing an y mutation detection strategy. In the case of somatic mutations in the gene encoding the tumor suppressor, p53, a clinically useful mutation detection assay must be able to detect a wide variety of types of mutations scattere d over five coding exons and their flanking intron sequences. This study ex amined the ability of a mutation detection strategy, termed NIRCA(TM), to i dentify single-base mutations in the clinically relevant domain of the p53 gene. This strategy relies on RNase digestion-mediated cleavage of double-s tranded copy RNA transcribed in vitro from polymerase chain reaction (PCR)- amplified genomic templates to detect mismatched base pairs resulting from hybridization of complimenting mutant and wild-type copy RNA strands. This assay system was found to robustly detect all twelve possible mismatches an d the plus one and minus one frame shifts. Furthermore, the assay could det ect mutations in clinical specimens when the mutant alleles composed as few as 4% of the total population of alleles isolated in bulk specimen genomic DNA. This mutation detection strategy worked efficiently in bladder, breas t, colon and lung tumors as well as sediments from bladder cytology specime ns.