Uchihi R, Yamamoto T, Kojima T, Tamaki K, Katsumata Y
Department of Legal Medicine, Nagoya University School of Medicine, Japan.
Nihon Hoigaku Zasshi. 1994 Oct;48(5):329-35.
A semi-nested polymerase chain reaction (PCR) was introduced to amplification of the HLA-DQA1 gene, which is a single-copy gene, in a single genome. The limitation of template DNA for genotyping is 1 ng of genomic DNA, or more in the case of ordinary PCR. When reamplification with the same primers was performed, primer dimer was generated and the sensitivity was not improved. We designed a semi-nested primer for the second round of PCR, using the semi-nested PCR, more than 3 pg of template DNA could be amplified and typed. Furthermore, this method was applied to amplify DQA1 gene in single human sperm having haploid DNA, and followed by typing with sequence-specific oligonucleotide (SSO) probes. The semi-nested PCR technique was found to enhance the sensitivity of the amplification reaction and allowed the successful typing of the HLA-DQA1 gene. This is helpful for genotyping from samples with extremely small amounts of DNA, such as forensic or ancient DNA samples.
采用半巢式聚合酶链反应（PCR）对单拷贝基因HLA - DQA1在单倍体基因组中进行扩增。基因分型时模板DNA的限制量为1 ng基因组DNA，普通PCR时所需模板量更多。当使用相同引物进行再次扩增时，会产生引物二聚体，且灵敏度并未提高。我们设计了用于第二轮PCR的半巢式引物，采用半巢式PCR，可扩增并分型3 pg以上的模板DNA。此外，该方法还应用于扩增单倍体DNA的单个人类精子中的DQA1基因，随后用序列特异性寡核苷酸（SSO）探针进行分型。结果发现，半巢式PCR技术可提高扩增反应的灵敏度，并成功实现HLA - DQA1基因的分型。这有助于对极少量DNA样本进行基因分型，如法医或古DNA样本。
