SINGLE-STRAND CONFORMATIONAL POLYMORPHISM USING CAPILLARY ELECTROPHORESIS WITH 2-DYE LASER-INDUCED FLUORESCENCE DETECTION

Capillary electrophoresis (CE), using a replaceable polymer matrix and two-dye laser-induced fluorescence has been applied to single strand conformational polymorphism (SSCP). Two-dye laser-induced fluorescence has been used for improved strand identification over a single-dye approach. Conditions suitable in the capillary format for rapid separation and high resolution have been explored. The influence of separation parameters such as temperature and matrix composition on separation in SSCP was first determined. Short analysis times allowed for fast screening of optimal separation conditions of the sample. Based on these results, the two strands of a standard 255 bp fragment of the lacI gene were resolved within 25 min with replaceable linear polyacrylamide as a separation matrix. The method was then applied to the detection of different mutations, in the presence of wild type, of a 276 bp fragment of the insulin-like growth factor 1-binding protein 1 (IGF1-BP3) gene.