Molecular Biotechnology

, Volume 32, Issue 2, pp 111–116 | Cite as

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

  • K. V. Satyanarayana
  • A. Chandrashekar
  • 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 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.

Index Entries

PCR genome walking ligation-mediated PCR uneven PCR promoter 


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Copyright information

© Humana Press Inc 2006

Authors and Affiliations

  • K. V. Satyanarayana
    • 1
  • A. Chandrashekar
    • 1
  • G. A. Ravishankar
    • 1
  1. 1.Plant Cell Biotechnology DepartmentCentral Food Technological Research Institute (CFTRI)KarnatakaIndia

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