Ligation-mediated PCR of restriction fragments from large DNA molecules.

Abstract

A general method is described for PCR amplification of single restriction fragments from large DNA molecules. The method involves sequence-specific ligation of synthetic oligonucleotides to ambiguous 4-base 5' overhangs produced by type IIS restriction endonucleases. Such "adapter-tags" provide one target for primer annealing in subsequent PCR reactions. The second target for primer annealing is provided by a universal "bubble-tag" ligated to blunt ends produced with another endonuclease. The key advantage of this approach is that specific fragments can be isolated without any prior knowledge of the nucleotide sequence of the target. Using bacteriophage lambda DNA as a test system, unique PCR products could be generated consistently. Conditions of temperature, ionic strength, and substrate concentration in the adapter-tag ligations--which affect sequence specificity--were found to have a major influence on the purity of PCR-generated fragments. In principle, the method permits the amplification of virtually any sequence from purified cosmid or YAC DNA using a library of only 240 adapter-tags.