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Multiplex single nucleotide extension: a robust and high throughput method for HLA-A locus typing.

Mei Han·Yue Qing Tan·Yanzheng Zhang·Jeannette Tsai·Rob Vorhaben·J Roberto Moraes·M Elisa Moraes·Peter Stastny
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Abstract

This report describes a typing method that can identify all known human leukocyte antigen A (HLA-A) alleles by determining nucleotides present at polymorphic sites using single nucleotide extension. Allele specific primers are bound to capture oligonucleotides which allows for a multiplex approach during single nucleotide extension (SNE) reactions. Eleven group-specific polymerase chain reaction amplifications were performed to obtain the templates to be analyzed with sets of primers designed to investigate the polymorphisms. Extension of biotin-labeled ddNTPs onto allele-specific primers was catalyzed by a DNA polymerase and each primer was hybridized to a specific capture oligonucleotide covalently bound to a bead. After staining with streptavidin-PE, incorporated fluorescence was determined with a flow cytometer. Fluorescence intensities were interpreted by computer and the nucleotide sequence was translated into HLA-A genotypes. Group-specific amplification reactions and primer sets for SNE were validated with 42 reference samples of known HLA-A alleles. In addition, 296 samples from three populations (N. A. Caucasian, African-American, Terena S. A. Indian) were analyzed and results compared to previous typing by SSOP. Reproducibility between repeated typings was 100% and ambiguities were quite rare. The method has been found to be accurate, relatively simple to perform and fast. It is our method of choice for high resolution clinical HLA-A typing.

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AllelesBase SequenceHLA-A AntigensHistocompatibility TestingHumansLeukocytesOligonucleotide ProbesPolymerase Chain ReactionPolymorphism, Single-Stranded ConformationalReproducibility of ResultsJournal Article

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Mei Han, Yue Qing Tan, Yanzheng Zhang, Jeannette Tsai, Rob Vorhaben, J Roberto Moraes, M Elisa Moraes, Peter Stastny. 2003. Multiplex single nucleotide extension: a robust and high throughput method for HLA-A locus typing.. https://doi.org/10.1016/j.humimm.2003.08.358

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