Fragment walking for long DNA sequencing by using a library as small as 16 primers

A DNA sequence can be rapidly and efficiently determined by digesting it into segments small enough to be sequenced at one time, then assembling them into contigs by searching for overlaps. Fragment walking achieves this without subcloning or preparing many kinds of primers; fragments obtained by digesting a template DNA are sequenced in parallel directly from the fragment mixture by using a set of 16 primers. Since the sequence adjacent to each cutting site is determined together with the fragment sequence, the contiguous fragment can be easily determined. The complete template DNA sequence can thus efficiently determined. The sequencing of pUC19 (2.7 kb) from both sides was done in one step by using only 15 fragments. The redundancy was only about 1.3, greatly reducing base-reading redundancy. Automating this strategy would increase the speed and efficiency of large-scale DNA sequencing.