Solid-phase direct DNA sequencing of allele-specific polymerase chain reaction-amplified HLA-DR genes

Abstract

We describe in this report a new strategy to directly sequence polymerase chain reaction-amplified human leucocyte antigen DRB genes using biotinylated allele-specific synthetic oligonucleotide primers coupled to streptavidin-coated magnetic beads. The use of allele-specific primers in the polymerase chain reaction allows for selective amplification of DNA from one haplotype, which when combined with the direct sequencing technique circumvents the need for DNA cloning prior to sequencing. We demonstrate here that this method can be used to characterize human leucocyte antigen DRB genes among heterozygous individuals. This method can be used for the rapid analysis of highly polymorphic genes among individuals heterozygous at the gene of interest.