Screening of Mutations/SNPs through Spectrophotometric Melt-Curve Analysis: A Preliminary Study on Non-Electrophoretic and No-Dye Method of Mutation Detection

Abstract

Presently available methods for screening of mutation require florescent labeled probe/dye or electrophoresis or both that make these methods tedious, expensive and time consuming. Here we described a method of screening of mutation/SNP without need of probe/dye and electrophoresis. The technique requires only PCR product and spectrophotometer with peltier. We screened mutation by monitoring DNA melting profile and transition temperature of homoduplex and heteroduplex by recording absorbance of UV by melting duplex. Absorbance for each duplex was measured at 260 nm from 60°C to 85°C at an increment of 1°C temperature using spectrophotometer having peltier with a heating rate of 1°C /min. In the heteroduplex samples there is rapid increase in the absorbance of UV at transition temperatures of 70°C. While in homoduplex sample it is reached after 75°C. Mutation in the sample was detected by observing the decrease of transition temperature of heteroduplex samples compared to homoduplex.