Yasuda T, Nadano D, Tenjo E, Takeshita H, Sawazaki K, Nakanaga M, Kishi K
Department of Legal Medicine, Fukui Medical School, Japan.
Electrophoresis. 1995 Oct;16(10):1889-93. doi: 10.1002/elps.11501601310.
We recently completely elucidated the molecular basis of genetic polymorphism in human deoxyribonuclease I and found it to be controlled by four codominant alleles, DNASE1*1, *2, 3 and 4. In this paper we describe a novel DNase I-genotyping system that could be used directly on DNA samples using the polymerase chain reaction (PCR) based on the three nucleotide substitutions underlying the protein polymorphism. The system consists of three independent reactions. Since the substitutions neither suppress nor create any known enzyme recognition site in the DNase I gene, two separate mismatched PCR followed by XhoI digestion methods were introduced to discriminate between the DNASE11 (or 3) and the DNASE12 (or 4) alleles, and to detect the DNASE14 allele. An amplification refractory mutation system was employed to detect DNASE13. A 100% correlation was found between the results of this genotyping method and those obtained by phenotyping using conventional isoelectric focusing. The high sensitivity and specificity of this genotyping method allows us to survey DNase I-polymorphism in small DNA samples.
我们最近彻底阐明了人类脱氧核糖核酸酶I基因多态性的分子基础，发现它受四个共显性等位基因DNASE11、2、3和4控制。在本文中，我们描述了一种新型的DNase I基因分型系统，该系统可基于蛋白质多态性背后的三个核苷酸替换，利用聚合酶链反应（PCR）直接对DNA样本进行操作。该系统由三个独立反应组成。由于这些替换在DNase I基因中既不抑制也不产生任何已知的酶识别位点，因此引入了两种单独的错配PCR随后进行XhoI消化的方法，以区分DNASE11（或3）和DNASE12（或4）等位基因，并检测DNASE14等位基因。采用扩增阻滞突变系统检测DNASE13。该基因分型方法的结果与使用传统等电聚焦进行表型分析所获得的结果之间存在100%的相关性。这种基因分型方法的高灵敏度和特异性使我们能够在小DNA样本中检测DNase I多态性。
