A novel human leucocyte antigen‐DRB1 genotyping method based on multiplex primer extension reactions

Abstract

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.

Finnish HLA studies confirm the increased risk conferred by HLA-B27 homozygosity in ankylosing spondylitis

“…17 Genotyping of HLA-DRB1 and HLA-B HLA-DRB1 genotyping was carried out using either direct sequencing (see the appendix for details), the primer extension method developed in this laboratory, 18 or the polymerase chain reaction-sequence specific primer (PCR-SSP) approach. 19 All the probands (n = 261) were genotyped using both the primer extension method and either direct sequencing or the PCR-SSP; the consistency between the genotypes was checked.…”

Non-B27 MHC associations of ankylosing spondylitis

“…DRB1 subtyping was performed to the resolution of the classical DRB1*01-10 alleles using the SNaPshot method. 63 Nineteen SNPs were identified within the 1-Mb region between TNF and DRB1, with an average inter-marker distance of 35 kb. Genotyping was carried out by matrixassociated laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry 64 or restriction fragment length polymorphism.…”

Real-Time PCR Using Fluorescent Resonance Emission Transfer Probes for HLA-B Typing

“…Our typing method may be favorably compared with other previously described PCR-based methods [8,9,12,13,15,37] because it can substantially reduce some labor-intensive steps and the total processing time. By skipping post-PCR steps, we can obtain typing results in less than 65 minutes and reduce the risk of sample contamination because the amplified products are always inside a lid-lock tube.…”

“…In this report we describe a real-time PCR-based assay that avoids some of the major drawbacks of currently available protocols for HLA-B PCR genotyping [8,9,12,13,15,37].…”