John J. Dunn, Laura-Li Butler-Loffredo and F. William Studier
Biology Department, Brookhaven National Laboratory, Upton, New York 11973
Primer walking using oligonucleotides selected from a library is an attractive strategy for large-scale DNA sequencing. Strings of three adjacent hexamers can prime DNA sequencing reactions specifically and efficiently when the template is saturated with a single stranded DNA-binding protein (1), and a library of all 4,096 hexamers is manageable. We would like to be able to sequence directly on 35-kbp fesmid templates, but the signal from a single round of synthesis is relatively weak and triple-hexamer priming has not yet been adapted for cycle sequencing. We reasoned that a hexamer library might be used for cycle sequencing if combinations of hexamers could be selectively ligated by using other hexamers as the template for alignment. In this way, the longer primers needed for cycle sequencing could be generated easily and economically without the need for complex machines for de novo synthesis.
We found that ordered ligation of 3 hexamers to form an 18-mer occurs readily on a template of the 3 complementary hexamers (offset by three base pairs) that can base pair unambiguously to form a double-stranded complex of indefinite length (2). Each hexamer forms three complementary base pairs with two other hexamers, generating complementary chains of contiguous hexamers with strand breaks staggered by three bases. Two adjacent hexamers in the chain to be ligated contain 5' phosphate groups and the others are unphosphorylated. Both T4 and T7 DNA ligase can ligate the phosphorylated hexamers to their neighbors in such a complex at hexamer concentrations in the 50-100 µM range, producing an 18-mer and leaving three unphosphorylated hexamers. The products of these ligation reactions can be used directly for fluorescent cycle sequencing of 35-kbp templates.
Unambiguous ligation requires that alternative complexes with perfect base pairing not be possible with the combination of hexamers used. Since the combination of hexamers is dictated by the sequence of the desired ligation product, some oligonucleotides cannot be produced unambiguously by this method. However, 82.5% of all possible 18-mers could potentially be generated starting with a library of all 4096 hexamers, more than adequate for high throughput DNA sequencing by primer walking.
Supported by the Office of Health and Environmental Research of the U. S. Department of Energy.
(1) Kieleczawa, J., Dunn, J. J., and Studier, F. W. DNA sequencing by primer walking with strings of contiguous hexamers. Science, 258, 1787-1791 (1992).
(2) Dunn, J. J., Butler-Loffredo, L. and Studier, F. W. Ligation of hexamers on hexamer templates to produce primers for cycle sequencing or the polymerase chain reaction. Anal. Biochem. 228, 91-100 (1995)