Garrity P A, Wold B J
Division of Biology, California Institute of Technology, Pasadena 91125.
Proc Natl Acad Sci U S A. 1992 Feb 1;89(3):1021-5. doi: 10.1073/pnas.89.3.1021.
We have developed a simplified procedure for the ligation-mediated polymerase chain reaction (LMPCR) using Thermococcus litoralis DNA polymerase (Vent DNA polymerase). We show that Vent DNA polymerase produces correct, blunt-ended primer extension products with substantially higher efficiency than Thermus aquaticus (Taq) DNA polymerase or modified T7 DNA polymerase (Sequenase). This difference leads to significantly improved genomic sequencing, methylation analysis, and in vivo footprinting with LMPCR. These improvements include representation of all bands with more uniform intensity, clear visualization of previously difficult regions of sequence, and reduction in the occurrence of spurious bands. It also simplifies the use of DNase I cut DNA for LMPCR footprinting.
我们开发了一种使用嗜热栖热放线菌DNA聚合酶（Vent DNA聚合酶）进行连接介导的聚合酶链反应（LMPCR）的简化程序。我们发现，Vent DNA聚合酶产生正确的平端引物延伸产物的效率明显高于水生栖热菌（Taq）DNA聚合酶或修饰的T7 DNA聚合酶（测序酶）。这种差异使得LMPCR在基因组测序、甲基化分析和体内足迹分析方面有显著改进。这些改进包括所有条带的强度更均匀、以前难以测序的区域能清晰显现以及假带出现频率降低。它还简化了用于LMPCR足迹分析的经DNA酶I切割的DNA的使用。
