Satyanarayana K V, Chandrashekar A, Ravishankar G A
Plant Cell Biotechnology Department, Central Food Technological Research Institute (CFTRI), Mysore, Karnataka 575020, India.
Mol Biotechnol. 2006 Feb;32(2):111-6. doi: 10.1385/MB:32:2:111.
Several polymerase chain reaction (PCR)-based methods are available for isolation of unknown genomic fragments. In the present study, a comparative evaluation of a few methods of ligation-mediated PCR methods and a ligation-independent one were made by isolating promoter fragment for N-methyltransferase gene involved in the caffeine biosynthetic pathway of Coffea canephora. The benefits of tertiary PCR and the effects of a 4-base cutting restriction endonuclease on the size of the PCR products obtained were demonstrated in one of the ligation-mediated PCR methods. The methods adopted in this study differed in the sizes of the 5'-flanking regions obtained. The efficiencies of various methods used reflect the inherent limitations of the PCR-based methods for isolation of unknown flanking regions.
有几种基于聚合酶链反应（PCR）的方法可用于分离未知的基因组片段。在本研究中，通过分离参与卡内弗拉咖啡咖啡因生物合成途径的N-甲基转移酶基因的启动子片段，对几种连接介导的PCR方法和一种不依赖连接的方法进行了比较评估。在其中一种连接介导的PCR方法中，证明了三级PCR的优点以及一种4碱基切割限制内切酶对所得PCR产物大小的影响。本研究采用的方法在获得的5'侧翼区域大小方面存在差异。所使用的各种方法的效率反映了基于PCR的方法在分离未知侧翼区域方面的固有局限性。
