COOPERATIVITY OF HEXAMER LIGATION

The SPEL-6 (Sequential rimer elongation by ligation of 6-mers) procedu re is based on the assembly of DNA primers by ligation of three or mor e hexamers taken from a library of 4096 hexamers. In this way, the syn thesized primers enable DNA sequencing by primer walking. Ligation by both T4 DNA ligase and Rhodothermus marinus thermophilic DNA ligase is highly cooperative. Sequencing ladders obtained with 18-60-nucleotide (nt) primers (produced by ligation of three to ten hexamers using T4 DNA ligase) were all of high quality, with no spurious bands. R. marin us DNA ligase requires at least seven hexamers for successful primer s ynthesis. Long primers (up to 60 nt), which are easy to obtain, especi ally by automated ligation, offer a definite advantage in DNA priming in regions with pronounced secondary structure. Moreover, the SPEL-6 p rocedure for DNA sequencing reduces the sequencing effort manifold. An additional application of hexamer ligation is the detection of point mutants, as described here.