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The advent of genomics-based technologies has revolutionized many fields of biological enquiry. However, chromosome walking or flanking sequence cloning is still an important needed tool to isolate the unknown DNA sequences flanked by known sequences or T-DNA insertions in molecular biology research. In this article, we described and compared the principles, efficiency, and applications of the five reported technically less complex methods for chromosome walking or flanking sequence cloning, namely (i) thermal asymmetric interlaced PCR (TAILPCR), (ii)high-efficiency TAILPCR(hiTAIL-PCR),(iii)fusion primer and nested integrated-PCR (FPNIPCR),(iv) Sitefinding-PCR and (v)Selfformed Adaptor PCR (SEFAPCR).While they are all based on the randomly primed PCR without enzyme digestion and ligation procedures, they show difference in efficiency and applications due to the different principles. Methodology The principle, efficiency, and applications of the five enzyme digestion-free methods:(i) TAIL-PCR, (ii) hiTAIL-PCR,(iii) FPNI-PCR, (iv) SiteFinding-PCR and (v) SEFA-PCR are reviewed in detail. Comparative analysis is undertaken to illustrate that the newly developed FPNI-PCR method has shown to be more powerful, effective, time-saving, and accurate than the established conventional reported methods. Conclusion Randomly primed PCR methods represent simplicity, less complex manipulations and possibly will be continuously widely employed by many molecular biology research laboratories. The newly developed FPNI-PCR can be done in a shorter time-scale and it is more powerful, effective, and accurate than conventional TAIL-PCR and will be widely used for genome walking and flanking sequence cloning.

Randomly primed and ligation-independent PCR methods: High efficiency and cost effective strategies for genome walking and flanking sequence cloning