High-throughput class I HLA genotyping using fluorescence resonance energy transfer (FRET) probes and sequence-specific primer-polymerase chain reaction (SSP-PCR).
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.
Gelsthorpe
A R
AR
Cancer and Immunogenetics Laboratory, Imperial Cancer Research Fund, Institute of Molecular Medicine, John Radcliffe Hospital, Oxford, UK.
Wells
R S
RS
Lowe
A P
AP
Tonks
S
S
Bodmer
J G
JG
Bodmer
W F
WF
D017931:DNA Primers; D005456:Fluorescent Dyes; D015395:Histocompatibility Antigens Class I / Q000235:genetics* / Q000276:immunology*; D006650:Histocompatibility Testing / Q000379:methods*; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods; D012680:Sensitivity and Specificity; D013050:Spectrometry, Fluorescence
D017931:DNA Primers; D005456:Fluorescent Dyes; D015395:Histocompatibility Antigens Class I