A RNA/RNA heteroduplex cleavage analysis to detect rare mutations in populations

• Published in: Molecular ecology 1997-07, Vol.6 (7), p.687-690
• Main Authors: Lenk, P., Wink, M.
• Format: Article
• Language: English
• Subjects: Animals; Base Sequence; Cytochrome b Group - genetics; DNA Primers; DNA, Mitochondrial - chemistry; DNA, Mitochondrial - genetics; DNA-Directed RNA Polymerases; Emys orbicularis; Molecular Sequence Data; Mutation; Nucleic Acid Heteroduplexes - metabolism; Polymerase Chain Reaction - methods; Ribonucleases; RNA - metabolism; Transcription, Genetic; Turtles - genetics
• ISSN: 0962-1083; 1365-294X
• DOI: 10.1046/j.1365-294X.1997.00233.x

In order to quantify genetic differences or gene flow within or between populations rare mutations need to be detected. In principle, the amplification and sequencing of marker genes is an appropriate method. However, because large numbers of individuals have to be screened, DNA sequencing is often too time consuming and too expensive. Therefore, alternative methods which combine good resolution with simplicity and economy are still in demand. In search for a quick, reliable, and nonisotopic alternative to study the phylogeography of the European pond turtle Emys orbicularis we have chosen a technique (`nonisotopic RNAse Cleavage Assay', NIRCA) developed by AMBION. The AMBION method [based on previous findings of Myers et al. (1985) and Winter et al. (1985)] exploits the properties of RNAse A to detect and cleave single mismatched bases in RNA/RNA heteroduplex molecules. Although originally designed for detecting single base substitutions, we describe here its additional use for multimismatches (up to 10) in RNA/RNA heteroduplexes.