[Blood-group genotyping by constant denaturing gel electrophoresis (CDGE)].

Abstract

Constant Denaturing Gel Electrophoresis (CDGE) was used as an analytical method of the genetic markers. Even a single base change in a fragment amplified by PCR was detected exactly by CDGE. The computational simulation of CDGE gave the calculation whether a single base change in a fragment amplified by PCR could be detected by CDGE or not. In this report, genotyping of three blood groups, MN, Duffy and Kidd, and Gc system is described. The regions reflecting allelic differences of each system were amplified from genomic DNAs. The concentration of denaturants (urea and formamide) in CDGE gels was decided with the computational simulation as follows: 15% for MN, 27% for Duffy, 24% for Kidd, 30% for Gc. CDGE was run in TBE buffer at 60 degrees C, 100 V constant voltage. PCR amplified fragments with 1-3 base changes were separated clearly in each gel. By staining the gels with ethidium bromide, the genotype of each system was determined. The genotyping of system by CDGE can avoid mistakes in the conventional method, which requires complicated and multiple troublesome operations. Analysis of PCR amplified fragments by CDGE will make a beneficial contribution to medico-legal practice.