Fluorescence-based sequencing of double-stranded DNA by hexamer string priming

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)

URL: http://www.ncbi.nlm.nih.gov/pubmed/8921192
DOI: 10.1006/abio.1996.0404

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 Deposited: 25 Apr 2013 15:21
Last Modified: 25 Apr 2013 15:21
Related URLs:
URI: http://repository.cshl.edu/id/eprint/28212

Actions (login required)

Administrator's edit/view item Administrator's edit/view item
CSHL HomeAbout CSHLResearchEducationNews & FeaturesCampus & Public EventsCareersGiving