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High throughput HLA sequence-based typing (SBT) utilizing the ABI Prism 3700 DNA Analyzer
S D Adams1, K C Barracchini, T B Simonis
1Department of Transfusion Medicine, Warren G Magnuson Clinical Center, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892-1184, USA.
Aims And Background:
The genetic complexity of the human major histocompatibility complex (MHC) has required the development of various molecular typing methods. The purpose of this paper is to compare the results of two of these molecular methods: sequenced based typing (SBT) and polymerase chain reaction (PCR) using sequence specific primers (PCR-SSP).
Methods:
The SBT method described utilizes an ABI Prism 3700 DNA Analyzer, which has been designed fro high throughput production of sequence data through highly automated operation with significant walk-away time. The ABI Prism 3700 DNA Analyzer is a 96-capillary electrophoresis instrument with the capability of running four 96-well plates black to back in a sixteen-hour period. Potentially, data from this machine can produce Class I sequences for A or B loci for 64 samples in this time frame. The SBT method encompassed exons 2, 3, and 4 with forward and reverse sequence orientation reactions using the PE Biosystems HLA-A and HLA-B Sequenced Based Typing Kits (PE Applied Biopsystems/Perkin-Elmer, Foster City, CA, USA). Most SBT methods previously employed only gather data from exons 2 and 3 which distinguishes most of the polymorphism necessary to identify the majority of alleles in the HLA region. However, in an effort to discern numerous null alleles in the HLA region, exon 4 data is also included. The PCR-SSP method utilized consists of one 96 well tray, with 95 primer mixes and one negative control, per sample designed to produce an intermediate/high resolution HLA-A, B typing.
Results:
Data from one 96-well capillary run on the ABI Prism 3700 DNA Analyzer, which consists of results from 16 samples for HLA-A or HLA-B loci, was compared to data derived from sixteen HLA-A and HLA-B PCR-SSP typings. 75% of loci tested achieved a higher resolution HLA typing by the SBT method.
Discussion:
The ability to provide allele level HLA typing results can have significant functional implications for the bone marrow transplant community and numerous vaccine studies.