Fluorescence-based sequencing of double-stranded DNA by hexamer string priming.
A fluorescence-based, T7 (Sequenase) dye terminator method for sequencing double-stranded DNA using strings of three contiguous hexamers as primers and single-stranded binding protein is described. In this method, the circular, supercoiled DNA vector pUC19 is first linearized with a restriction enzyme to create a sequenceable template. Sequencing is then accomplished using three cycles of "denaturation," annealing, and extension/termination. Twenty-two of 33 hexamer strings tested in a controlled study produced acceptable sequence, with read lengths varying from the mid 300s to the low 400s and a base-calling accuracy of at least 97%. To test its potential utility in directed DNA sequencing, the protocol was then used to completely sequence both strands of pUC19. For this test project, a total of 28 hexamer strings was used with an overall successful priming rate of 75%. The current protocol appears to be sufficiently robust to be used in the finishing phase of a shotgun sequencing project and is amenable to semiautomation. Prospects for using the protocol for full-scale directed sequencing as well as for full automation are discussed.
Johnson
A F
AF
Lita Annenberg Hazen Genome Center, Cold Spring Harbor Laboratory, New York 11724-0100, USA.
Lodhi
M A
MA
McCombie
W R
WR
D001331:Automation; D001483:Base Sequence; D004247:DNA; D005456:Fluorescent Dyes; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D017422:Sequence Analysis, DNA
D005456:Fluorescent Dyes; D004247:DNA