Nagai M, Yoshida A, Sato N
Department of Biochemistry, Iwate Medical University School of Dentistry, Japan.
Biochem Mol Biol Int. 1998 Jan;44(1):157-63. doi: 10.1080/15216549800201172.
The effects of bovine serum albumin, dithiothreitol, and glycerol on PCR were studied. PCR under standard conditions failed the amplification of an enterohemorrhagic E. coli DNA fragment when the boiled bacterial cell lysate was used as the template. The addition of either one of bovine serum albumin, dithiothreitol, or glycerol in the reaction mixture allowed the specific fragment amplification; and the optimum concentrations were as follows: bovine serum albumin, 1 mg/ml; dithiothreitol, 10 mM; and glycerol, 5%. In addition, when all of the three agents were included at the above concentrations, the PCR yield was further increased. The effect of the three-agent mixture was not the template specific. Furthermore, the mixture enabled long PCR over 20 kb when Taq DNA polymerase with 3'-5' exonuclease activity was used for the amplification. Our simple PCR method allows robust PCR independent of template purity or amplification length.
研究了牛血清白蛋白、二硫苏糖醇和甘油对聚合酶链反应（PCR）的影响。当使用煮沸的细菌细胞裂解物作为模板时，在标准条件下进行的PCR未能扩增出肠出血性大肠杆菌DNA片段。在反应混合物中添加牛血清白蛋白、二硫苏糖醇或甘油中的任何一种都能实现特异性片段扩增；最佳浓度如下：牛血清白蛋白，1mg/ml；二硫苏糖醇，10mM；甘油，5%。此外，当以上述浓度同时包含这三种试剂时，PCR产量进一步提高。三种试剂混合物的效果并非模板特异性的。此外，当使用具有3'-5'外切核酸酶活性的Taq DNA聚合酶进行扩增时，该混合物能够实现超过20kb的长片段PCR。我们简单的PCR方法能够实现稳健的PCR，而与模板纯度或扩增长度无关。
