27 - Identification of Rhodopsin Gene Mutations Using GC-Clamped Denaturing Gradient Gel Electrophoresis

Abstract

Publisher Summary Several techniques have been developed that make it possible to detect single base changes in DNA. These techniques include allele-specific oligonucleotide (ASO) hybridization, RNase cleavage, chemical cleavage, single-stranded conformation polymorphism (SSCP) analysis, heteroduplex analysis, and denaturing gradient gel electrophoresis (DGGE). Some of these techniques are not suitable for screening DNA for mutations. GC-clamped DGGE makes it possible to detect nearly 100% of single-base changes in DNA fragments of about 500 bp in length. This chapter describes the use of GC-clamped DGGE for the detection of rhodopsin coding sequence mutations. These methods are also applicable to the study of other candidate genes that may be involved in human retinal degeneration. DGGE can physically separate fragments differing by a single-base substitution on the basis of a difference in melting temperature (T m ) of the two molecules. A single-base change alters the T m of a given DNA melting domain by up to 2°C. Melting of a domain changes the conformation of the DNA molecule and, thus, affects its rate of migration through a polyacrylamide gel matrix.