High-throughput class I HLA genotyping using fluorescence resonance energy transfer (FRET) probes and sequence-specific primer-polymerase chain reaction (SSP-PCR)

Tissue Antigens. 1999 Dec;54(6):603-14. doi: 10.1034/j.1399-0039.1999.540611.x.

Abstract

We have developed a semi-automated HLA class I typing system utilising TET/TAMRA-labelled fluorescence resonance energy transfer (FRET) hydrolysis probes. The results from 87 individuals are in full concordance with serology and conventional gel-based systems. This assay replaces labour-intensive conventional gel-based DNA typing and has a higher allelic resolution than serology. Our approach differs from previously published fluorogenic probe typing protocols in that it provides simultaneous typing of HLA-A, -B and -C loci to medium resolution. Furthermore, by using equipment that is not specific to FRET probe analysis our system has in-built expansion capacity to 384 reactions per plate, thus making it applicable to high-throughput population screening. Automation is achieved through the use of computer software which analyses direct input from the fluorescence reader, allowing high throughput with a low inherent error rate.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • DNA Primers
  • Fluorescent Dyes
  • Histocompatibility Antigens Class I / genetics*
  • Histocompatibility Antigens Class I / immunology*
  • Histocompatibility Testing / methods*
  • Humans
  • Polymerase Chain Reaction / methods
  • Sensitivity and Specificity
  • Spectrometry, Fluorescence

Substances

  • DNA Primers
  • Fluorescent Dyes
  • Histocompatibility Antigens Class I