High-throughput single-base mismatch detection for genotyping of UDP-glucuronosyltransferase (UGT1A1) with probe capture assay coupled with modified allele-specific primer extension reaction (MASPER)

Abstract

We have developed a new method based on specific primer extension reactions coupled with plate hybridization for high-throughput genotyping of single-base mutations. To improve the switching characteristics of the primer extension reaction, we introduced an artificial mismatch two bases upstream of the 3'-terminal base in the detection primers. A set of primers that correspond to wild-type and mutant DNA segments can be used to accurately analyze single-base mutations. The termini of these primers are at the mutation positions. The primer extension products produced by polymerase chain reaction (PCR) were captured by an oligonucleotide probe immobilized on the surface of microtiter wells and were detected by a colorimetric assay using the streptavidin-conjugated horseradish peroxidase. We used the new method to genotype 96 individuals for 211G>A (G71R) and 119 for 1456T>G (Y486D) in the UDP-glucuronosyltransferase1A1 gene; the results were completely concordant with those found by direct sequencing. The proposed method includes ordinary PCR and a microplate assay format, and may be used in routine laboratory tests.