High-throughput single-strand conformation polymorphism analysis by automated capillary electrophoresis: Robust multiplex analysis and pattern-based identification of allelic variants

Genetic diagnosis of an inherited disease or cancer often involves analysis for unknown point mutations in several genes; therefore, rapid and automat ed techniques that can process a large number of samples are needed. We des cribe a method for high throughput single strand conformation polymorphism (SSCP) analysis using automated capillary electrophoresis. The operating te mperature of a commercially available capillary electrophoresis instrument (ABI PRISM 310) was expanded by installation of a cheap in-house designed c ooling system, thereby allowing us to perform automated SSCP analysis at 14 -45 degrees C. We have used the method for detection of point mutations ass ociated with the inherited cardiac disorders long QT syndrome (LQTS) and hy pertrophic cardiomyopathy (HCM). The sensitivity of the method was 100% whe n 34 different point mutations were analyzed, including two previously unpu blished LQTS-associated mutations (F157C in KVLQT1 and G572R in HERG), as w ell as eight novel normal variants in HERG and MYH7, The analyzed polymeras e chain reaction (PCR) fragments ranged in size from 166 to 1,223 bp. Seven teen different sequence contexts were analyzed. Three different electrophor esis temperatures were used to obtain 100% sensitivity Two mutants could no t be detected at temperatures greater than 20 degrees C. The method has a h igh resolution and good reproducibility and is very robust, making multiple x SSCP analysis and pattern-based identification of known allelic variants as single nucleotide polymorphisms (SNPs) possible. These possibilities, co mbined with automation and short analysis time, make the method suitable fo r high-throughput tasks, such as genetic screening. Hum Mutat 13:318-327, 1 999. (C) 1999 Wiley-Liss, Inc.