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

Research output: Contribution to journalArticlepeer-review

4 Scopus citations

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.

Original languageEnglish (US)
Pages (from-to)1111-1122
Number of pages12
JournalHuman Immunology
Volume64
Issue number12
DOIs
StatePublished - Dec 2003

Keywords

  • A*6817
  • Automation
  • Flow cytometry
  • Microspheres
  • Terena

ASJC Scopus subject areas

  • Immunology and Allergy
  • Immunology

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