Abstract
An enhanced universal fast walking (UFW) method adapted for the mapping of transposons is described. This protocol combines the original UFW method with the use of agarase to unravel composite nucleotide sequence, thereby forgoing molecular cloning steps and the use of restriction enzymes and ligases necessary in other available genome walking methods such as the prominent inverse PCR. The minuscule automatable chemistry of UFW is completed within one reaction vessel using a constant enzyme buffer, and the intrinsic DNA fingerprints, from which amplicons may be quantitatively recovered, offer quality assurance. The core steps of the protocol, spanning half a day or less, comprise first-strand synthesis, primer destruction, random-ended-primer annealing, distal branched-end repair, second-primer destruction, lariat formation and final amplification. Distinctively, no starting or intermediate templates are wasted during the reaction series, thus achieving yields comparable to direct PCR. Ultimate per-reaction walk-lengths are schematically illimitable and sequence-ready amplicons can be produced immediately from prevalent single-copy genomic walk origins. The core UFW protocol may be applied, as described here, to expedited transposon boundary retrieval, but is also applicable to general genome walking and cDNA walking, as well as viral and other insertional element mapping.
