The specific aim of this study is to prove the feasibility of the investigators' SPEL-6 (sequential primer elongation employing ligation of 6-mers) method for the progressive sequencing of 7- to 50-kb DNA fragments by primer walking, and thus to develop sequencing techniques which will be at least 10-100 times more efficient than present methods. The SPEL-6 method permits the investigators to synthesize 18-mers (or 24-mers) in only 10 minutes by ligation of hexamers on the sequenced template in the presence of ligase, ATP, and enabling proteins, e.g., SSB (single-strand binding protein of Escherichia coli), and then immediately producing very high quality sequencing ladders in the standard dideoxy sequencing reaction. Their SPEL-6 procedure was 100 percent successful with sequences tested; moreover, quality and reproducibility of the sequence ladders is much superior to those obtained with nonligated hexamers, as reported recently. Ligation of hexamers (available as a library) makes primer walking a very powerful sequencing method, very amenable to automating. The investigators' next objective is to totally automate the SPEL-6 primer walking procedures, as to permit sequencing a 50- to 100-kb DNA fragment in 1-2 weeks; that means that the entire E. coli genome could be sequenced in 2 weeks when a 50-channel SPEL-6 sequencer is fully developed. This is thought feasible, since the investigators routinely produce extremely clear and sharp 300- to 500-nt ladders with the 18-mer primer, which was ligated from hexamers. Almost every element of the automated primer walking will have to be optimized, including (a) conversion of 50- to 100-kb fragments (phage lambda DNA serves as a model) to the single-stranded (and anchored) configuration; (b) selecting the most suitable ligase (efficient at low temperature), SSB or other enabling protein, and DNA polymerase; (c) determining the minimal size for a hexamer library (between 500 and 4,096 hexamers), partially based on the investigators' observation that 18-mers with one mismatch (=17- mers) are still quite efficient primers under proper annealing conditions; (d) developing automation for the (i) computerized hexamer design, as based on last 50 nt of just determined ca. 500 nt sequence, (ii) rapid synthesis of 18-mers by ligation of hexamers selected from the library, (iii) ultrathin layer (capillary) sequencing with the ligated 18-mer primers, and (iv) rapid readout of the fluorescent bands and sequence assembly. The investigators' long-term objective is to make sequences of large (50-100 kb) fragments very rapid, while avoiding a need for any subcloning in M13 vectors, and for any random bottom-up assembly of sequence contigs. Thus, their SPEL-6 primer walking may make the human genome project much more feasible, and also would permit its rapid application as a diagnostic tool for many clinical syndromes.
