van Dijk A, Melchers R, Tilanus M, Rozemuller E
Department of Immunology, University Medical Centre Utrecht, Utrecht, The Netherlands.
Tissue Antigens. 2007 Apr;69 Suppl 1:64-5. doi: 10.1111/j.1399-0039.2006.760_5.x.
The use of direct sequencing as a typing strategy is well acknowledged. Direct sequencing identifies all sequence motifs including new polymorphisms in heterozygous sequences. The earlier protocols for human leukocyte antigen HLA-DQB1 Sequencing-Based Typing (SBT) frequently encounter preferential amplification of one of the alleles that can lead to unreliable sequences or even to allelic dropout. In our new approach, the quality of the exon 2 sequences, now including both alleles to the same extend, was achieved by amplifying the HLA-DQB1*05/06 group into two groups by changing the common 3' amplification primer. In combination with exon 3 this updated HLA-DQB1 protocol provides a reliable approach for heterozygous sequencing.
直接测序作为一种分型策略的应用已得到广泛认可。直接测序可识别所有序列基序，包括杂合序列中的新多态性。早期用于人类白细胞抗原HLA - DQB1基于测序的分型（SBT）方案经常遇到一个等位基因的优先扩增，这可能导致不可靠的序列，甚至等位基因缺失。在我们的新方法中，通过改变通用的3'扩增引物将HLA - DQB1*05/06组扩增为两组，从而在相同程度上实现了外显子2序列（现在包括两个等位基因）的质量。结合外显子3，这种更新后的HLA - DQB1方案为杂合子测序提供了一种可靠的方法。
