Nozawa H, Yamamoto T, Uchihi R, Yoshimoto T, Tamaki K, Hayashi S, Ozawa T, Katsumata Y
Department of Legal Medicine, Nagoya University School of Medicine, Nagoya 466-8550, Japan.
Leg Med (Tokyo). 1999 Apr;1(2):61-7. doi: 10.1016/s1344-6223(99)80014-5.
The typing of nuclear DNA from hair shafts has often been unsuccessful to date. We tried to type one of the nuclear DNA loci, HLA-DQA1, from hair shafts, using an efficient cetyl-trimethyl ammonium bromide (CTAB) precipitation for DNA purification and a sensitive semi-nested PCR. After thorough washing with ethanol and water, hair shafts were digested by proteinase K in the presence of dithiothreitol, followed by a purification step including CTAB-DNA precipitation. The specific region of HLA-DQA1 gene was amplified by the semi-nested PCR, and the amplified products were cloned and sequenced. The HLA-DQA1 genotype was determined by comparing the sequence to the known sequence of each allele. All genotypes of HLA-DQA1 were successfully typed with hair shafts from six known heterozygotes, although one of them showed the predominant appearance of one allele. For correct typing, a template DNA equivalent to a hair shaft of 5 or 10 cm in length was necessary. Without the CTAB-DNA precipitation step, DNA extract from such hair shafts inevitably contains enough melanin to inhibit PCR. The present results suggest that hair shafts can be used for the typing of nuclear DNA loci.
迄今为止，从毛发干中进行核DNA分型往往并不成功。我们尝试使用高效的十六烷基三甲基溴化铵（CTAB）沉淀法纯化DNA，并采用灵敏的半巢式PCR，从毛发干中对一个核DNA位点HLA - DQA1进行分型。用乙醇和水充分洗涤后，毛发干在二硫苏糖醇存在的情况下用蛋白酶K消化，随后进行包括CTAB - DNA沉淀的纯化步骤。通过半巢式PCR扩增HLA - DQA1基因的特定区域，并对扩增产物进行克隆和测序。通过将序列与每个等位基因的已知序列进行比较来确定HLA - DQA1基因型。来自六个已知杂合子的毛发干成功完成了所有HLA - DQA1基因型的分型，尽管其中一个显示出一个等位基因占主导的情况。为了进行正确的分型，需要相当于5或10厘米长毛发干的模板DNA。如果没有CTAB - DNA沉淀步骤，来自此类毛发干的DNA提取物不可避免地含有足够的黑色素来抑制PCR。目前的结果表明，毛发干可用于核DNA位点的分型。
