Yoshii T, Tamura K, Taniguchi T, Akiyama K, Ishiyama I
Department of Legal Medicine, Teikyo University School of Medicine, Tokyo, Japan.
Nihon Hoigaku Zasshi. 1993 Aug;47(4):323-9.
It has been confirmed that water-soluble eumelanins often extracted together with DNAs from natural black hairs act as an inhibitor of Taq DNA polymerase in the polymerase chain reaction (PCR). In the present investigation, an attempt to amplify the non-coding 333-bp region of mitochondrial DNA (mt333DNA) produced the following results: 1) Water-soluble preparations made from chemically synthesized melanin (Sigma products), as well as natural black eumelanins, inhibited the PCR amplification of mt333DNA at concentrations of more than 2 micrograms/ml. 2) Quantitative measurement of Taq DNA polymerase-catalyzed DNA synthesis in terms of the amount of [alpha-32P] dCMP incorporated into activated calf thymus DNA showed that both of the water-soluble melanins had the same inhibition activity as represented by the sigmoidal curve derived from a quadratic equation of melanin concentration. This observation suggested that Taq DNA polymerase combined with two molecules of melanin to form an inactivated complex. 3) Melanins did not appear to affect either the thermostability of Taq DNA polymerase at 94 degrees C, or the step of primer-annealing to template DNAs. On the other hand, we established a simple and useful method for removal of water-soluble eumelanins contaminating DNA preparations from hairs. The method was based on the adsorption of melanins to Bio-Gel. When a Bio-Gel P-60 minicolumn was equilibrated with 10 mM sodium acetate buffer, pH 4.2, water-soluble melanins were completely adsorpted to it whereas DNAs passed through, although the melanins showed incomplete adsorption to the gel when it was equilibrated with TE (10 mM Tris-HCl, pH 7.5, 0.1 mM EDTA).(ABSTRACT TRUNCATED AT 250 WORDS)
现已证实，常与天然黑发中的DNA一起提取的水溶性真黑素在聚合酶链反应（PCR）中可作为Taq DNA聚合酶的抑制剂。在本研究中，对线粒体DNA（mt333DNA）非编码333bp区域进行扩增时得到了以下结果：1）由化学合成黑色素（Sigma产品）以及天然黑色真黑素制成的水溶性制剂，在浓度超过2微克/毫升时会抑制mt333DNA的PCR扩增。2）根据掺入活化小牛胸腺DNA中的[α-32P] dCMP量对Taq DNA聚合酶催化的DNA合成进行定量测量，结果表明两种水溶性黑色素具有相同的抑制活性，其抑制活性由黑色素浓度二次方程得出的S形曲线表示。该观察结果表明Taq DNA聚合酶与两分子黑色素结合形成失活复合物。3）黑色素似乎既不影响Taq DNA聚合酶在94℃时的热稳定性，也不影响引物与模板DNA的退火步骤。另一方面，我们建立了一种简单且有用的方法来去除毛发DNA制剂中污染的水溶性真黑素。该方法基于黑色素对Bio-Gel的吸附。当Bio-Gel P-60微型柱用pH 4.2的10 mM醋酸钠缓冲液平衡时，水溶性黑色素会完全吸附在上面，而DNA则会通过，不过当用TE（10 mM Tris-HCl，pH 7.5，0.1 mM EDTA）平衡时，黑色素对凝胶的吸附不完全。（摘要截短于250字）
