SAITOH Hisako Chiba University, School of Mecicine, Research Assistant, 医学部, 助手 (10292674)
SATO Yayoi Chiba University, School of Mecicine, Research Assistant, 医学部, 助手 (70009679)
KUROSAKI Kunihiko Tokyo Medical College, Assistant Professor, 医学部, 助教授 (60240701)
|Budget Amount *help
¥3,200,000 (Direct Cost : ¥3,200,000)
Fiscal Year 1999 : ¥1,000,000 (Direct Cost : ¥1,000,000)
Fiscal Year 1998 : ¥2,200,000 (Direct Cost : ¥2,200,000)
HLA-DR, -DQB locus have been known to be high polymorphic in the HLA ClassII region, so it seems to be useful tool for forensic identification. Then the hot start polymerase chain reaction with sequence specific primers (Hot Start PCR-SSP) method is superior to the other PCR-based method at the point of the rapidness, simplicity and reproducibility.
In the present study, we used the Hot Start PCR-SSP method to type HLA-DR from forensic biological materials and consider the availability to the forensic routine work. As materials, a total of 172 samples (bloodstains, saliva stains, hairs, cigarette butts, vaginal swabs, nail, bone and tooth) were collected from Japanese volunteers and the autopsied bodies. We used a commercial available primer kit. DNA from those samples except for cigarette butts was extracted with phenol/chloroform and purified by the Microcon (R)100 microconcentrator. Hot Start PCR was performed following the SSP set manual with slight modification. We used AmpliTaq(R)
Gold and Microcon(R)100 microconcentrator to perform hot start PCR and to purify and concentrate the extracts, respectively. PCR amplification was undergone by 35〜40 cycles after incubation at 94℃ for 10minutes. The alleles were identified by the 2% agarose gel electrophoresis prestained with ethidium bromide in the presence of specific PCR products. The typing could be performed within approximately 4 hours after DNA extraction. We could perform the typing from DNA as little as 10 pg. in the PCR reaction.
PCR amplifications could be performed from 161 out of 172 samples (94%). The rate of successful typing from bloodstains, saliva stains, hairs, cigarette butts, vaginal swabs and nail was 66/68 (97%), 38/38 (100%), 6/7 (86%), 52/56 (93%), 2/2 (100%) and 1/1 (100%), respectively. These types corresponded to the known types of the donors identified from the liquid blood. We also tried to type from the aged degraded DNA and could type 5 out of 7 samples extracted from 10- and 20-year-old bloodstains. The failure contained both false negative and false positives. One of these causes was supposed to be too little template DNA. The contamination and inhibitors in the sample may also induce the failure.
These results indicate that majority of samples is able to type repidly from various forensic materials. It has been known that the PCR-SSP method had a disadvantage to need many PCR reactions and was considered not to be suitable for the little and degraded samples, however, it was improved to increase the sensitivity using the hot start PCR technique and a purification device. We conclude the method is suitable to the forensic routine work because of the rapidness, simplicity and reproducibility. Less