Specific-primer-directed DNA sequencing using automated fluorescence detection

Automated fluorescence-based DNA sequence analysis offers the possibility to undertake very large scale sequencing projects. Directed strategies, such as the specific-primer-directed sequencing approach ('gene walking'), should prove useful in such projects. Described herein is a study inv...

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Bibliographic Details
Published in:Nucleic acids research 1989-08, Vol.17 (15), p.6087-6102
Main Authors: KAISER, R. J, MACKELLAR, S. L, VINAYAK, R. S, SANDERS, J. Z, SAAVEDRA, R. A, HOOD, L. E
Format: Article
Language:English
Subjects:
Analytical, structural and metabolic biochemistry
Autoanalysis
automated methods
Base Sequence
Biological and medical sciences
Biotechnology
Chromatography, High Pressure Liquid
Cloning, Molecular
DNA
Fluorescent Dyes
Fundamental and applied biological sciences. Psychology
Genetic engineering
Genetic technics
Methods. Procedures. Technologies
Molecular Sequence Data
Nucleic acids
Nucleic bases, nucleotides
Oligonucleotides - chemical synthesis
Software
Synthetic digonucleotides and genes. Sequencing
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Summary:Automated fluorescence-based DNA sequence analysis offers the possibility to undertake very large scale sequencing projects. Directed strategies, such as the specific-primer-directed sequencing approach ('gene walking'), should prove useful in such projects. Described herein is a study involving the use of this approach in conjunction with automated fluorescence detection on a commercial instrument (ABI 370A DNA sequencer). This includes procedures for the rapid chemical synthesis and purification of labeled primers, the design of primer sequences that are compatible with the commercial analysis software, and automated DNA sequence analysis using such primers. A set of four fluorophore-labeled primers can be reliably synthesized in a twenty-four hour period, and greater than 300 nucleotides of analyzed new sequence obtained using this set in an additional twenty-four hours. Scale-up of these procedures to take advantage of the full capabilities of the sequencer is, at present, too slow and costly to be suitable for routine sequencing, and therefore the use of specific-primers is best suited to the closure of gaps in extended sequence produced using random cloning and sequencing strategies.
ISSN:0305-1048
1362-4962
DOI:10.1093/nar/17.15.6087