Simultaneous analysis of mutant and normal alleles for multiple cystic fibrosis mutations by the ligase chain
1Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, Texas 77030-3498, USA.
Abstract:
The ligase chain reaction (LCR) involves repetitive cycles of ligation of two adjacent pairs of oligonucleotides to form longer ligated products in a template-dependent manner. This study demonstrates the application of LCR for analysis of multiple small mutations. We adapted the technology for the simultaneous determination of the normal and mutant alleles in a competition format, as well as multiple mutations in a multiplex format. For these purposes, we used mutations causing cystic fibrosis, namely the delta F508, W1282X, and G551D mutations. Blunt ligation was compared to a strategy with a single base gap on one or both strands to be filled by thermostable polymerase prior to ligation. Blunt or gap strategies worked well for detection of the delta F508 mutation. Detection of the W1282X mutation worked well with a blunt strategy when high K+ concentration (180-220 mM) was used to reduce template-independent ligation. For reliable detection of the G551D mutation, we used mismatches in the oligonucleotides 2-5 bp away from the ligation site and hot start of the reaction to achieve allele specificity. Excellent discrimination of mutations was achieved using competitive LCR with six oligonucleotides (two common on one side of the mutation plus two wild type and two mutant on the opposite side with the mutation site at the end adjacent to the common oligonucleotides) and with multiplex-competitive LCR using 12 oligonucleotides to detect both alleles for two mutations in a single tube.(ABSTRACT TRUNCATED AT 250 WORDS)
