Island hopping--a method to sequence rapidly very large fragments of DNA

摘要

The present invention is a technique which allows one to determine rapidly the nucleic acid sequence of large fragments of nucleic acids such as the inserts obtained from YACs, BACs and Pls. This method uses an array of random primers matched pairwise in all combinations to amplify portions of the fragments to be sequenced. Some of these PCR reactions result in the formation of single bands of amplified DNA which are called islands. These islands are randomly scattered along the fragment of nucleic acid. These individual islands are sequenced, but this leaves major gaps in the complete sequence of DNA. A second round of PCR is performed in which the ends of the islands are used to design primers pointing away from the islands, these primers being matched pairwise in all combinations. This round of PCR again results in some of the reactions forming single bands of amplified nucleic acid. These bands connect the islands determined earlier. This automatically allows one to place the islands and connecting bands in order and to complete the sequencing of the gaps. Using this method obviates the necessity of subcloning portions of the fragment or slowly sequencing along the fragment in serial fashion and is therefore much more rapid and less labor intensive than previously used methods.