HLA-B typing by allele separation followed by direct sequencing

M. Eberle, L. A. Knapp, K. K. Iwanaga, M. J. Domanico, K. Aiyer, David Watkins

Research output: Contribution to journalArticle

17 Citations (Scopus)

Abstract

Due to the enormous allelic diversity of the HLA-B locus, it has been difficult to design an unambiguous molecular typing method for the alleles at this locus. Here we describe a technique for the direct sequencing of HLA-B alleles. Initially, HLA-B alleles were PCR-amplified after locus-specific reverse transcription of RNA. Alleles were then separated using denaturing gradient gel electrophoresis (DGGE), which separates DNA fragments based on their sequence composition. Amplification products were excised from the gel and eluted DNA was reamplified and directly sequenced. The derived sequences were aligned to a database of published HLA-B sequences, and an initial allele assignment was made. This approach was theoretically sufficient to type 92 of the 118 known HLA-B alleles. The majority of the remaining 26 alleles contain differences at the beginning of exon 2, a region outside the DGGE-separated PCR products. Therefore, we used heterozygous sequencing of this region to identify 19 of these 26 alleles, raising the resolution power to 111 alleles. Using this technique, we analyzed immortalized cell lines and blood samples from several different sources. Nine immortalized cell lines were obtained from the 10th International Histocompatibility Workshop (IHWS) and nine were derived from aboriginal peoples. Additionally, 25 blood samples were acquired from a panel of donors previously shown to be difficult to type using serological techniques. Altogether, using this new method of allele separation by DGGE followed by direct sequencing, we typed 52 different alleles from 57 individuals, covering 40 serological specificities.

Original languageEnglish
Pages (from-to)365-375
Number of pages11
JournalTissue Antigens
Volume49
Issue number4
StatePublished - May 6 1997
Externally publishedYes

Fingerprint

Histocompatibility Testing
HLA-B Antigens
Alleles
Denaturing Gradient Gel Electrophoresis
Molecular Typing
Cell Line
Polymerase Chain Reaction
Histocompatibility
DNA
Reverse Transcription
Exons
Gels
Databases
RNA

Keywords

  • cDNA
  • DGGE
  • HLA-B
  • PCR
  • Sequencing
  • Typing

ASJC Scopus subject areas

  • Immunology
  • Cell Biology

Cite this

Eberle, M., Knapp, L. A., Iwanaga, K. K., Domanico, M. J., Aiyer, K., & Watkins, D. (1997). HLA-B typing by allele separation followed by direct sequencing. Tissue Antigens, 49(4), 365-375.

HLA-B typing by allele separation followed by direct sequencing. / Eberle, M.; Knapp, L. A.; Iwanaga, K. K.; Domanico, M. J.; Aiyer, K.; Watkins, David.

In: Tissue Antigens, Vol. 49, No. 4, 06.05.1997, p. 365-375.

Research output: Contribution to journalArticle

Eberle, M, Knapp, LA, Iwanaga, KK, Domanico, MJ, Aiyer, K & Watkins, D 1997, 'HLA-B typing by allele separation followed by direct sequencing', Tissue Antigens, vol. 49, no. 4, pp. 365-375.
Eberle M, Knapp LA, Iwanaga KK, Domanico MJ, Aiyer K, Watkins D. HLA-B typing by allele separation followed by direct sequencing. Tissue Antigens. 1997 May 6;49(4):365-375.
Eberle, M. ; Knapp, L. A. ; Iwanaga, K. K. ; Domanico, M. J. ; Aiyer, K. ; Watkins, David. / HLA-B typing by allele separation followed by direct sequencing. In: Tissue Antigens. 1997 ; Vol. 49, No. 4. pp. 365-375.
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