High-throughput single-strand conformation polymorphism analysis on a microfabricated capillary array electrophoresis device.

ABSTRACT

A high-density 384-lane microfabricated capillary array electrophoresis device is evaluated for high-throughput single-strand conformation polymorphism (SSCP) analysis. A delayed back bias direct electrokinetic injection scheme is used to provide better than 10-bp resolution with an 8.0-cm effective separation length. Separation of a HaeIII digest of PhiX174 yielded theoretical plate numbers of 4.0 x 10(6). Using 5% PDMA containing 10% glycerol and 15% urea, 21 single-nucleotide polymorphisms (SNPs) from HFE, MYL2, MYL3, and MYH7 genes associated with hereditary hemochromatosis (HHC) and hereditary hypertrophic cardiomyopathy (HCM) are discriminated at two running temperatures (25 degrees C and 40 degrees C), providing 100% sensitivity. The data in this study demonstrate that the 384-lane microCAE device provides the resolution and detection sensitivity required for SSCP analysis, showing its potential for ultrahigh-throughput mutation detection.