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

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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.