Abstract
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.
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Satyanarayana, K.V., Chandrashekar, A. & Ravishankar, G.A. Evaluation of PCR-based methods for isolating flanking regions of genes. Mol Biotechnol 32, 111–116 (2006). https://doi.org/10.1385/MB:32:2:111
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DOI: https://doi.org/10.1385/MB:32:2:111