Denaturing gradient gel electrophoresis

A rapid method for differentiating BoLA-DRB3 alleles

B. M. Aldridge, S. M. McGuirk, R. J. Clark, L. A. Knapp, David Watkins, D. P. Lunn

Research output: Contribution to journalArticle

26 Citations (Scopus)

Abstract

The products of the BoLA-DRB3 locus are important molecules in the bovine immune response. Several techniques have been used to study and define this locus but they are generally time consuming and limited in their ability to detect novel alleles. In this study we used denaturing gradient gel electrophoresis (DGGE), and direct sequencing, for BoLA-DRB3-typing. First, modified locus-specific primers were used in polymerase chain reaction (PCR) to amplify a 240 bp fragment of exon 2 of BoLA-DRB3 from the genomic DNA of 22 cattle and one pair of twin calves. The reverse primer included a GC-rich clamp to improve the physical separation of the BoLA-DRB3 alleles by DGGE. The denaturing gradient needed to produce separation of alleles was determined using perpendicular DGGE, and this gradient was then applied to parallel denaturing gels. The optimal time for producing allele separation was determined using a time-series analysis. The bands representing individual BoLA-DRB3 alleles were excised from the gels, reamplified, and the nucleotide sequence determined using fluorescent-based automated cycle sequencing. The nucleotide sequences of the separated bands were then compared to published BoLA-DRB3 alleles. A gradient of 10-15% acrylamide combined with a 15-50% ureaformamide gradient was successfully used to separate BoLA-DRB3 alleles in all individuals examined. Nucleotide sequencing showed that the 24 animals possessed 13 BoLA-DRB3 alleles, all of which have been previously described. The BoLA-DRB3 genotypes included 20 heterozygotes and two homozygotes. Three BoLA-DRB3 alleles were seen in each of the twin calves, possibly due to leukochimerism. The technique is reliable and rapid, and avoids cloning alleles prior to nucleotide sequencing and therefore offers distinct advantages over previous techniques for BoLA-DRB3-typing.

Original languageEnglish
Pages (from-to)389-394
Number of pages6
JournalAnimal Genetics
Volume29
Issue number5
DOIs
StatePublished - Oct 1 1998
Externally publishedYes

Fingerprint

Denaturing Gradient Gel Electrophoresis
denaturing gradient gel electrophoresis
rapid methods
Alleles
alleles
loci
nucleotides
BoLA-DRB3 antigen
gels
calves
Nucleotides
Gels
nucleotide sequences
acrylamides
cattle
Acrylamide
homozygosity
Homozygote
Heterozygote
exons

Keywords

  • BoLA-DRB3
  • Bovine lymphocyte antigen
  • Denaturing gradient gel electrophoresis
  • Major histocompatibility complex

ASJC Scopus subject areas

  • Animal Science and Zoology
  • Genetics
  • Genetics(clinical)

Cite this

Denaturing gradient gel electrophoresis : A rapid method for differentiating BoLA-DRB3 alleles. / Aldridge, B. M.; McGuirk, S. M.; Clark, R. J.; Knapp, L. A.; Watkins, David; Lunn, D. P.

In: Animal Genetics, Vol. 29, No. 5, 01.10.1998, p. 389-394.

Research output: Contribution to journalArticle

Aldridge, B. M. ; McGuirk, S. M. ; Clark, R. J. ; Knapp, L. A. ; Watkins, David ; Lunn, D. P. / Denaturing gradient gel electrophoresis : A rapid method for differentiating BoLA-DRB3 alleles. In: Animal Genetics. 1998 ; Vol. 29, No. 5. pp. 389-394.
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