Johnson, A. F., Lodhi, M. A., McCombie, W. R. (1996) Fluorescence-based sequencing of double-stranded DNA by hexamer string priming. Analytical Biochemistry, 241 (2). pp. 228-237. ISSN 00032697 (ISSN)
Abstract
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.
| Item Type: | Paper |
|---|---|
| Uncontrolled Keywords: | double stranded dna article automation dna determination dna sequence dna structure fluorescence priority journal structure analysis Base Sequence DNA Fluorescent Dyes Molecular Sequence Data Polymerase Chain Reaction Sequence Analysis, DNA |
| Subjects: | bioinformatics bioinformatics > genomics and proteomics > genetics & nucleic acid processing > DNA, RNA structure, function, modification bioinformatics > genomics and proteomics > genetics & nucleic acid processing bioinformatics > genomics and proteomics Investigative techniques and equipment |
| CSHL Authors: | |
| Communities: | CSHL labs > McCombie lab |
| Depositing User: | Matt Covey |
| Date: | 1996 |
| Date Deposited: | 25 Apr 2013 15:21 |
| Last Modified: | 25 Apr 2013 15:21 |
| Related URLs: | |
| URI: | https://repository.cshl.edu/id/eprint/28212 |
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