PCR-Based Genome Walking 111 MOLECULAR BIOTECHNOLOGY Volume 32, 2006 RESEARCH 111 Molecular Biotechnology © 2006 Humana Press Inc. All rights of any nature whatsoever reserved. 1073–6085/2006/32:2/111–116/$30.00 *Author to whom all correspondence and reprint requests should be addressed. Plant Cell Biotechnology Department, Central Food Techno- logical Research Institute (CFTRI), Mysore, Karnataka, 575020, India. E-mail: pcbt@cscftri.ren.nic.in. Abstract Evaluation of PCR-Based Methods for Isolating Flanking Regions of Genes K. V. Satyanarayana, A. Chandrashekar, * and G. A. Ravishankar 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 meth- ods 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 demon- strated 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. Index Entries: PCR; genome walking; ligation-mediated PCR; uneven PCR; promoter. 1. Introduction The isolation of 5'-flanking regions of genes is an important step in the study of regulation of gene expression. Flanking regions of genes have been isolated conventionally by screening genomic libraries using cDNA probes; although efficient, this method consumes considerable amounts of time, effort, and expenses and normally requires the use of radioactivity (1). Since the introduc- tion of polymerase chain reaction (PCR) (2), sev- eral PCR-based methods for the isolation of flanking regions have been developed, and these methods are commonly referred to as “genome walking” (3). Inverse PCR (4) and ligation-medi- ated PCR (3,5,6) are some of the methods that rely on the presence of restriction sites in or near the target region. The restricted DNA fragments are self-ligated (intra-molecular ligation) to form circular molecules (inverse PCR) or ligated to DNA cassette/adapters (ligation-mediated PCR) prior to PCR. However, these techniques have sev- eral limitations and their high-failure rates may be attributed to dependence on the availability of fa- vorable restriction site around the known locus, circularization of DNA or reduced ligation effi- ciency between the cassette and restricted target DNA fragments, reduced annealing efficiency of cassette, mispriming, costs, and technical difficul- ties involved (3). Although several modifications of the cassette/adapters have been made (7–9), complete elimination of non-specific amplifica- tion is difficult. Ligation-independent methods are also avail- able wherein the restricted DNA fragments after denaturation are polyadenylated (10) or homo- polymer tailed (11) with terminal transferase. Techniques that are restriction- and ligation-indepen- dent include TAIL-PCR (12) and uneven PCR (13). However, the elaborate thermal cycling program in TAIL-PCR involving “supercycles” may be a