An automated fluorescent single strand conformation polymorphism techniquefor high throughput mutation screening

Objective To develope a high throughput mutational detection method by muti ple fluorescence-labeled polymerase chain reaction (PCR) products. Methods A total of 27 known mutations including 22 substitutions, 3 inserti ons (1, 2 and 7 bp) and 2 deletions (1 and 2 bp) in the hepatocyte nuclear factor (HNF)-4 alpha, glucokinase and HNF-1 alpha genes were tested. During nested PCR, amplified fragments were labeled with three fluorescent dyes. PCR products were visualized with an ABI-377 fluorescence sequencer using 5 % glycerol or 10% sucrose in nondenaturing gel conditions. Results Twenty-five of 27 variants (93%) could be detected by combining 5% glycerol and 10% sucrose gel matrix conditions. Twenty-two of 27 (82%) and 18 of 27 (67%) variants were identified using 5% glycerol and 10% sucrose c onditions, respectively. Conclusion This fluorescence-based PCR single strand conformation polymorph ism technique represents a simple, non-hazardous, time-saving and sensitive method for high throughput mutation detection.