[3]Simple and nonisotopic methods to detect unknown gene mutations in nucleic acids

Publisher Summary This chapter discusses simple and nonisotopic methods to detect unknown gene mutations in nucleic acids. Regardless of the method used to identify a gene, the demonstration of mutations that cosegregate with the disease phenotype is both an important step and an essential condition toward proving the involvement of the gene (product) in the pathological process. The principle of the method of single-strand conformation polymorphism (SSCP) analysis is that the electrophoretic mobility of single-stranded nucleic acid fragments under nondenaturing conditions depends largely on their conformation. The chapter presents a simple and nonisotopic method that can be carried out in any laboratory without special equipment and combines the SSCP and heteroduplex analyses. This method has been successfully used for mutation screening in 15 different genes responsible for inherited diseases. Because of the invention of highly selective DNA amplification by a thermostable DNA polymerase and repeated cycles of denaturation, primer annealing, and chain elongation, the polymerase chain reaction (PCR) has become one of the most widely used techniques in molecular genetics. The PCR provides fast and highly effective synthesis of any DNA (or cDNA) fragment specified by a pair of primers from a complex mixture of nucleic acids without time-consuming cloning procedures.