Method of solution-based scanning for alterations in a dna segment using a double-stranded dna binding dye and fluorescence melting profiles

Abstract

A method for determining whether a DNA sequence is identical to a wild-type sequence is presented. A GC-rich DNA segment is attached to a DNA segment of interest. With the GC-clamp attached, the DNA segment of interest has two melting domains, a higher domain associated with the GC-clamp and a lower domain associated with the DNA segment of interest. The DNA segment of interest is labeled with a fluorescent label such as a double-stranded DNA binding dye and mixed with a denaturant. The mixture of denaturant and fluorescently labeled DNA is heated. Fluorescence is monitored to determine the melting point of the DNA segment of interest. The melting temperature of the DNA segment of interest is compared to the melting point of the wild-type sequence. A difference in melting temperatures of the DNA sequence and the wild-type sequence indicates an alteration in the DNA sequence. In a presently preferred embodiment, some homozygous mutations may be better detected by combining approximately equal parts of the DNA segment of interest with equal parts of the wild-type sequence to create a heteroduplex. The heteroduplex is fluorescently labeled and mixed with a denaturant. The mixture is heated while monitoring fluorescence to determine the melting point of the heteroduplex. A difference in melting temperatures of the heteroduplex and the wild-type sequence indicates an alteration in the DNA sequence.