Denaturing gradient gel electrophoresis: a rapid method for differentiating BoLA‐DRB3 alleles

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.

Molecular characterization of expressed DQA and DQB genes in the California sea lion ( Zalophus californianus )

“…5). While this finding provided strong evidence for Zaca-DQ sequence homology between individuals, bands with similar migration patterns may have different sequences (Aldridge et al 1998). Each band was therefore extracted, reamplified and directly sequenced.…”

“…An adapted denaturing gradient gel electrophoresis (DGGE) (Aldridge et al 1998) was used to examine the degree ofZaca-DQ nucleotide variation between individuals ftom different geographical locations. Peripheral blood leukocytes were isolated by whole blood lysis of samples collected from wild-caught sea lions in the Sea of Cortez, Baja, Mexico (n=9), and from San Miguel Island, California, USA (n=10).…”

Asymmetric PCR-SSCP: a Useful Tool for Detection of OLA-DRB1(MHC Class Ii) Gene Polymorphism in Slovak Improved Valachian Sheep

“…Though the DNA sequencing is gold standard for most of the phylogenetic studies, the cost requisite for such analysis is quite high as well as it is time consuming. Recently, simple and rapid techniques like PCR-RFLP (restriction fragment length polymorphism) and DGGE (denaturing gradient gel electrophoresis) have been used by different researchers for the detection of MHC gene polymorphism (Aldridge et al 1998;Konnai et al 2003). These techniques enable to group the individuals into clusters on the basis of gene polymorphism.…”

Comparison ofHelicobacterspp. in Cheetahs (Acinonyx jubatus) with and without Gastritis

“…To determine if more than one type of Helicobacter organism was present within the stomachs of cheetahs, a fragment of the 16S rRNA gene was amplified from gastric samples with universal primers (244f and 528r from Table 1). A GC clamp was attached to the 5Ј end of the forward primer (CGCCCGCCGC GCCCCGCGCC CGGCCCGCCG CCGCCGCTAT GTCCTATCAG CTTGTTG) to improve separation of closely related sequences (1). PCR conditions were similar to those previously described, and amplified products were separated in a 12% polyacrylamide gel with a 20 to 60% gradient of urea-formamide (DCode electrophoresis reagent kit, Bio-Rad, Richmond, Calif.) run in TAE buffer at 60°C and 300 V for 4 h in a Bio-Rad D-Gene apparatus (Bio-Rad, Richmond, Calif.).…”