16 DNA Analysis Under Highly Denaturing Conditions in Bare Fused Silica Capillaries

Denaturing electrophoresis has widely been used for DNA sequencing and mutation detection. Specific electromigration behavior of completely or partially dissociated DNA molecules is the prerequisite of high-selective separations, based on their size and/or conformation differences. Various denaturing techniques have been developed for both slab gel (SGE) and capillary electrophoresis (CE) formats. The highest possible denaturing ability of the background electrolytes (BGE) of both the sample solution and electrophoresis buffer is used for sequencing, and for analyses of restriction fragment length polymorphism (RFLP), or for length polymorphism of fragments amplified by polymerase chain reaction (PCR-FLP) (1 –5 ). In the technique of single-strand conformation polymorphism (SSCP) analysis, completely denatured samples are loaded into a native sieving medium where ssDNA fragments adopt a conformation that is determined by their nucleotide sequence (6 ,7 ). Thus, not only complementary strands, but also strands carrying mutations are separated under optimum conditions. The SSCP technique is very sensitive, and even the point mutations, i.e., the substitutions of a single nucleotide in a sequence, can be detected. In the constant denaturant (CDCE) (8 ,9 ) or denaturing gradient capillary electrophoresis (DGCE) techniques (10 ,11 ), a native sample is loaded into a sieving medium with a moderate denaturing ability. Consequently, dsDNA molecules dissociate according to their melting temperature, which sensitively reflects the mutations in the DNA sequence. The optimum denaturing power of a BGE is controlled by the temperature (constant or programmed) of the run, and by the concentration of denaturing agent(s). Based on the extent of dissociation, mutant DNA sequences are separated reproducibly from wild-type sequences, with high resolution.