Evaluation of PCR-based methods for isolating flanking regions of genes.

Satyanarayana, K. V. and Chandrashekar, A. and Ravishankar, G. A. (2006) Evaluation of PCR-based methods for isolating flanking regions of genes. Molecular Biotechnology, 32. pp. 111-116. ISSN 1073-6085

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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.

Item Type:	Article
Uncontrolled Keywords:	Caffeine; Polymerase chain reaction; Coffea canephora
Subjects:	500 Natural Sciences and Mathematics > 07 Life Sciences > 03 Biochemistry & Molecular Biology 500 Natural Sciences and Mathematics > 10 Plants > 05 Tissue Culture 500 Natural Sciences and Mathematics > 07 Life Sciences > 02 Evolution & genetics
Divisions:	Plant Cell Biotechnology
Depositing User:	Users 16 not found.
Date Deposited:	01 Jun 2011 06:00
Last Modified:	01 Jun 2011 06:00
URI:	http://ir.cftri.res.in/id/eprint/1947

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