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  • Title: Evaluation of PCR-based methods for isolating flanking regions of genes.
    Author: Satyanarayana KV, Chandrashekar A, Ravishankar GA.
    Journal: Mol Biotechnol; 2006 Feb; 32(2):111-6. PubMed ID: 16444012.
    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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