Rozemuller E H, Bouwens A G, Bast B E, Tilanus M G
Diagnostic DNA Laboratory, Utrecht University Hospital, The Netherlands.
Hum Immunol. 1993 Aug;37(4):207-12. doi: 10.1016/0198-8859(93)90503-s.
Routine HLA typing by serology has been supported by DNA or protein analysis of the respective molecules in cases when serologic typing was inconclusive or difficult to perform. DNA analysis by RFLP, SSO, or PCR amplification with SSP is a reliable tool for the identification of alleles. Because DNA sequences may now be determined routinely, we developed software based on sequence data from known sequences for allele assignment of polymorphic gene systems. We describe the assignment of HLA-DPB alleles based upon sequence data obtained after PCR amplification and subsequent sequencing of exon 2. Our software includes a database containing all known HLA-DPB sequences, which offers the possibility of analyzing heterozygous individuals by combinatorial comparison through all alleles and thus identifying the one or two alleles involved. Quality control of the sequence has been included by the alignment of constant regions of the sequence combined with related polymorphism of known polymorphic nucleotides and the identification of the positions crucial for the allele assignment. The ability to type for HLA-DPB alleles routinely by automatic sequence determination and subsequent automated analysis offers new perspectives for HLA-DNA typing.
当血清学分型不确定或难以进行时,通过对相应分子进行DNA或蛋白质分析来支持常规的血清学HLA分型。通过RFLP、SSO或用SSP进行PCR扩增的DNA分析是鉴定等位基因的可靠工具。由于现在可以常规确定DNA序列,我们基于已知序列的序列数据开发了软件,用于多态基因系统的等位基因分配。我们描述了基于PCR扩增和随后外显子2测序获得的序列数据进行HLA - DPB等位基因分配的方法。我们的软件包括一个包含所有已知HLA - DPB序列的数据库,该数据库通过对所有等位基因进行组合比较来分析杂合个体,从而识别所涉及的一个或两个等位基因。通过将序列的恒定区与已知多态核苷酸的相关多态性进行比对以及确定对等位基因分配至关重要的位置,实现了序列的质量控制。通过自动序列测定和随后的自动分析来常规进行HLA - DPB等位基因分型的能力为HLA - DNA分型提供了新的前景。