A Simple and Efficient Method to Determine the Terminal Sequences of Restriction Fragments Containing Known Sequences

We present an improvement of the inverse PCR method for the determination of end sequences of restriction fragments containing unknown DNA sequences flanked by known segments. In this approach, a short "bridge" DNA is inserted during the self-ligation step of the inverse PCR technique. This bridge DNA acts as primer annealing sites for amplification and subsequent direct sequencing. Successive PCR amplifications enable selective amplification of the unknown sequences from a complex mixture. Unlike previously described methods, our method does not require special materials, such as synthetic adapters or biotinylated primers that must be prepared each time to adapt the target. Furthermore, no complex steps such as dephosphorylation or purification are needed. Our method can save time and reduce the cost of cloning unknown sequences; it is ideal for routine, rapid gene walking. We applied this method to a GC-rich bacterial genome and succeeded in determining the end sequences of a 4.5-kb fragment.