Aldridge B M, McGuirk S M, Clark R J, Knapp L A, Watkins D I, Lunn D P
Department of Medical Sciences, School of Veterinary Medicine, University of Wisconsin, Madison 53706, USA.
Anim Genet. 1998 Oct;29(5):389-94. doi: 10.1046/j.1365-2052.1998.295361.x.
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.
牛白细胞抗原-DRB3（BoLA-DRB3）基因座的产物是牛免疫反应中的重要分子。已经使用了几种技术来研究和定义该基因座，但它们通常耗时且检测新等位基因的能力有限。在本研究中，我们使用变性梯度凝胶电泳（DGGE）和直接测序对BoLA-DRB3进行分型。首先，使用修饰的基因座特异性引物进行聚合酶链反应（PCR），以从22头牛和一对双胞胎小牛的基因组DNA中扩增BoLA-DRB3外显子2的240 bp片段。反向引物包含一个富含GC的夹钳，以通过DGGE改善BoLA-DRB3等位基因的物理分离。使用垂直DGGE确定产生等位基因分离所需的变性梯度，然后将该梯度应用于平行变性凝胶。使用时间序列分析确定产生等位基因分离的最佳时间。从凝胶中切下代表单个BoLA-DRB3等位基因的条带，重新扩增，并使用基于荧光的自动循环测序确定核苷酸序列。然后将分离条带的核苷酸序列与已发表的BoLA-DRB3等位基因进行比较。10-15%丙烯酰胺与15-50%尿素甲酰胺梯度的组合成功地用于分离所有检测个体中的BoLA-DRB3等位基因。核苷酸测序表明，这24只动物拥有13个BoLA-DRB3等位基因，所有这些等位基因先前均已被描述。BoLA-DRB3基因型包括20个杂合子和2个纯合子。在每对双胞胎小牛中都发现了三个BoLA-DRB3等位基因，这可能是由于白细胞嵌合体。该技术可靠且快速，避免了在核苷酸测序之前克隆等位基因，因此与以前的BoLA-DRB3分型技术相比具有明显优势。
