The development of a new HLA-DNA typing method using DNA direct sequencing.

高度多态性的人白细胞抗原（HLA）与细胞免疫反应有关，在法医学领域具有重要的鉴定价值。迄今为止，HLA II类基因座在聚合酶链反应（PCR）扩增后，通过限制性内切酶或序列特异性寡核苷酸探针进行分型。这样的程序通常是复杂和耗时的。因此，直接测序扩增的DNA可以提供快速分型的方法。然而，法医标本如血迹、精液渍、唾液渍和毛发通常含有可能抑制PCR反应的污染物。在这项研究中，我们试图分析污染物的性质，并尽量减少这些物质的影响。在头发中，我们发现黑色素是一种有效的聚合酶链反应抑制剂，现在我们正试图将这种物质的影响降到最低。在血迹和唾液污渍中，我们试图控制污染物质的抑制作用，最终制定了DNA纯化和分型的改进程序。于是，我们开始用DNA测序仪读取HLA-DQB1基因的序列。

Highly polymorphic human leukocyte antigen (HLA), which is related to cellular immunoreaction, is useful for personal identification in the field of forensic medicine. To date, HLA class II loci are typed by restriction enzymes or sequence-specific oligonucleotide probes after being amplified by the polymerase chain reaction (PCR). Such procedures are usually complicated and time consuming. Therefore, direct sequencing of the amplified DNA could provide a rapid typing method. However, forensic specimens such as bloodstains, seminal stains, saliva stains and hairs usually contains contaminants which may inhibit PCR reaction. In this study, we tried to analyze the nature of the contaminants, and to minimize the effect of those substances. In hair, we found that melanin is a potent inhibitor for the PCR reaction, and are now trying to minimize the effect of this substances. In bloodstains and saliva stains, we tried to control the inhibition of the contaminated substances, and finally made an improved procedure of DNA purification and typing.So, we started to read the sequence of HLA-DQB1 gene with DNA sequencer.