From the double-helix to novel approaches to the sequencing of large genomes.

Elucidation of the structure of DNA by Watson and Crick [Nature 171 (1953) 737-738] has led to many crucial molecular experiments, including studies on DNA replication, transcription, physical mapping, and most recently to serious attempts directed toward the sequencing of large genomes [Watson, Science 248 (1990) 44-49]. I am totally convinced of the great importance of the Human Genome Project, and toward achieving this goal I strongly favor 'top-down' approaches consisting of the physical mapping and preparation of contiguous 50-100-kb fragments directly from the genome, followed by their automated sequencing based on the rapid assembly of primers by hexamer ligation together with primer walking. Our 'top-down' procedures totally avoids conventional cloning, subcloning and random sequencing, which are the elements of the present 'bottom-up' procedures. Fragments of 50-100 kb are prepared in sufficient quantities either by in vitro excision with rare-cutting restriction systems (including Achilles' heel cleavage [AC] or the RecA-AC procedures of Koob et al. [Nucleic Acids Res. 20 (1992) 5831-5836]) or by in vivo excision and amplification using the yeast FRT/Flp system or the phage lambda att/Int system. Such fragments, when derived directly from the Escherichia coli genome, are arranged in consecutive order, so that 50 specially constructed strains of E. coli would supply 50 end-to-end arranged approx. 100-kb fragments, which will cover the entire approx. 5-Mb E. coli genome. For the 150-Mb Drosophila melanogaster genome, 1500 of such consecutive 100-kb fragments (supplied by 1500 strains) are required to cover the entire genome. The fragments will be sequenced by the SPEL-6 method involving hexamer ligation [Szybalski, Gene 90 (1990) 177-178; Fresenius J. Anal. Chem. 4 (1992) 343] and primer walking. The 18-mer primers are synthesized in only a few minutes from three contiguous hexamers annealed to the DNA strand to be sequenced when using an over 100-fold excess of hexamers and T4 DNA ligase at room temperature, preferably in the presence of the single-strand-binding (SSB) protein of E. coli. These 18-nt primers are immediately extended by the DNA polymerase, Sequenase 2.0, in the dideoxy sequencing reaction. Very high quality sequencing ladders are obtained for single-stranded DNA or denatured double-stranded approx. 50-kb fragments, as exemplified by phage lambda DNA.(ABSTRACT TRUNCATED AT 400 WORDS)