PCR walking

PCR was originally designed for amplification of known DNA sequences, for which specific primers could be designed. This was possible, through the use of un cloned DNA, if amplification of DNA having known sequences is desired. However, when a fragment of DNA with unknown sequence needs to be amplified, this DNA fragment is cloned in a vector" so that the known sequences at the insertion site of the vector can be used as primers. More recently PCR methods have been designed, which allow amplification of DNA with unknown sequences, that are present adjoining a known sequence within a DNA fragment. The technique is described as PCR walking.

For instance, if cDNA sequence is known, the regulatory sequences adjoining the transcribed sequence in genomic DNA can be amplified. In this exercise, an adapter of known sequence is first ligated to the restriction fragments derived from genomic DNA, that contains the unknown sequence to be amplified. One of the two primers is designed from the known gene sequence (described as gene specific primer) and the other primer is designed on the basis of the adapter of known sequence, that is ligated to the restriction fragments of genomic DNA. Consequently, PCR walking involves the following three steps : (i) restriction enzyme digestion to produce a large number of genomic DNA fragments; (ii) ligation of adapters to restriction fragments and (iii) PCR amplification using one adapter primer and the other gene specific primer

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The adapter for the restriction fragments for PCR walking is designed to have an amine group on the 3' end of the lower smaller strand, to disallow extension of this end of the adapter, thus preventing generation of API primer binding site (this is a feature of a method described as 'vectorette PCR').

Further, the adapter primer is shorter than the adapter itself and binds to the end of adapter ligated fragments. 'The adapter also has inverted terminal repeats, so that if the PCR product contains double stranded adapter sequence at both ends (due to non-specific DNA synthesis), the ends of individual DNA strands will form 'panhandle' structures following every denaturation step; this suppresses their further amplification, thus permitting amplification of only those regions, which are adjacent to gene specific sequence, used for designing the second primer (this is called 'suppressor PCR'). The technique has been used for PCR walking upstream from exon of the human tissue-type plasminogen activator (tPA) gene, and upstream of the human transferrin receptor gene.