Katcher H L, Schwartz I
Department of Biochemistry and Molecular Biology, New York Medical College, Valhalla 10595.
Biotechniques. 1994 Jan;16(1):84-92.
The ability of thermostable DNA polymerases to mediate template-dependent DNA synthesis in the presence of phenol has been examined as monitored by amplification of a specific Borrelia burgdorferi rRNA sequence. Tth DNA polymerase displayed the unique property of maintaining both DNA- and RNA-dependent DNA polymerase activities in the presence of 2%-5% (vol/vol) of phenol-saturated PBS buffer. Tth DNA polymerase mediated reverse transcriptase activity was unaffected by phenol-saturated phosphate-buffered saline concentrations as high as 15% (vol/vol). By contrast, Taq DNA Polymerase was inactive under these conditions. The ability to function in the presence of phenol can greatly simplify reverse transcriptase, PCR and reverse transcription-PCR protocols since the phenol-saturated aqueous phase of a phenol partition can be added directly to the reaction mixtures. The simplicity of the procedures described should have applicability to a broad range of basic research, clinical and forensic applications.
通过对特定的伯氏疏螺旋体rRNA序列进行扩增监测，研究了热稳定DNA聚合酶在苯酚存在下介导模板依赖性DNA合成的能力。在2%-5%（体积/体积）苯酚饱和的PBS缓冲液存在下，Tth DNA聚合酶表现出维持DNA依赖性和RNA依赖性DNA聚合酶活性的独特特性。Tth DNA聚合酶介导的逆转录酶活性不受高达15%（体积/体积）的苯酚饱和磷酸盐缓冲盐水浓度的影响。相比之下，Taq DNA聚合酶在这些条件下无活性。在苯酚存在下发挥作用的能力可以极大地简化逆转录酶、PCR和逆转录-PCR方案，因为苯酚分配的苯酚饱和水相可以直接添加到反应混合物中。所描述程序的简单性应适用于广泛的基础研究、临床和法医应用。
