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We have developed and validated a semi-automated fluorescent method of genotyping human leucocyte antigen (HLA)-DRB1 alleles, HLA-DRB1*01-16, by multiplex primer extension reactions. This method is based on the extension of a primer that anneals immediately adjacent to the single-nucleotide polymorphism with fluorescent dideoxynucleotide triphosphates (minisequencing), followed by analysis on an ABI Prism 3700 capillary electrophoresis instrument. The validity of the method was confirmed by genotyping 261 individuals using both this method and polymerase chain reaction with sequence-specific primer (PCR-SSP) or sequencing and by demonstrating Mendelian inheritance of HLA-DRB1 alleles in families. Our method provides a rapid means of performing high-throughput HLA-DRB1 genotyping using only two PCR reactions followed by four multiplex primer extension reactions and PCR-SSP for some allele groups. In this article, we describe the method and discuss its advantages and limitations.

Type

Journal article

Journal

Tissue antigens

Publication Date

07/2004

Volume

64

Pages

88 - 95

Addresses

The Botnar Research Center, Oxford, UK.

Keywords

Humans, Spondylitis, Ankylosing, DNA Primers, HLA-DR Antigens, Fluorescent Dyes, Polymerase Chain Reaction, Base Sequence, Genotype, Polymorphism, Genetic, Polymorphism, Single-Stranded Conformational, Alleles, Finland, Female, Male