ASSEMBLY OF 18-NUCLEOTIDE PRIMERS BY LIGATION OF 3 HEXAMERS - SEQUENCING OF LARGE GENOMES BY PRIMER WALKING

A novel method (SPEL-6) for sequencing large genomes permits 10-min sy nthesis of 18-mer primers and their immediate use in DNA sequencing by primer walking. Primers are ligated from three contiguous hexamers co mplementary to the single-stranded or denatured DNA (150:1 ratio), whi ch is used both as the template for ligation and as the DNA to be sequ enced. The complete library consists of 4096 hexamers, but sequencing remains very efficient with a fourfold smaller library composed of 102 4 degenerate hexamers containing all four nucleotides at their positio n 3. The SSB protein (which binds to single-stranded DNA) greatly enha nces the quality of sequencing ladders. The SPEL-6 method eliminates t he need for subcloning, permits direct sequencing of large DNA fragmen ts (of 50 kb or larger), is ideally suited for automation, and should accelerate the sequencing of large genomes by more than one order of m agnitude. (C) 1994 Academic Press, Inc.