Automated DNA sequencing using 4-color fluorescent detection of reactions primed with hexamer strings

McCombie, W. R., Kieleczawa, J. (1994) Automated DNA sequencing using 4-color fluorescent detection of reactions primed with hexamer strings. Biotechniques, 17 (3). pp. 574-579. ISSN 07366205 (ISSN)

URL: http://www.ncbi.nlm.nih.gov/pubmed/7818912

Abstract

The use of strings of contiguous short primers rather than a single long primer has the potential to greatly diminish the cost, time and management efforts associated with sequencing by primer walking. For maximum impact, this chemistry must be adaptable to current fluorescent automated sequencers, which, while allowing automated acquisition of data, have reduced sensitivity when compared with radioactive-based sequencing. The ability to use hexamer strings on Applied Biosystems DNA sequencers has now been demonstrated for single-stranded templates. Procedures are described that allow sequence to be obtained up to 400 bases from the priming site. Signal strength is sufficient in this region to allow single base resolution and highly accurate automatic base calling to be performed by the sequencer. While these conditions can no doubt be further optimized, these results show the feasibility of inexpensive primer walking using hexamer string primers on currently available commercial DNA sequencers. This should have a wide range of applications from genome sequencing projects to the sequencing of cDNA clones without the necessity of creating nested deletions or the necessity of spending inordinate amounts of time and money on oligonucleotide synthesis.

Item Type: Paper
Uncontrolled Keywords: primer dna single stranded dna article dna sequence dna template fluorescence laboratory automation technique Base Sequence Molecular Sequence Data Sequence Analysis, DNA
Subjects: bioinformatics > genomics and proteomics > genetics & nucleic acid processing
bioinformatics > genomics and proteomics
Investigative techniques and equipment
bioinformatics > genomics and proteomics > genetics & nucleic acid processing > genomes
CSHL Authors:
Communities: CSHL labs > McCombie lab
Depositing User: Matt Covey
Date Deposited: 25 Apr 2013 20:33
Last Modified: 25 Apr 2013 20:33
Related URLs:
URI: http://repository.cshl.edu/id/eprint/28196

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