A Dye Terminator Method for Automated DNA Sequencing Using Four Fluorescent Dideoxynucleosides and Thermal Cycling

Automated DNA sequencers have been developed rapidly, but dye terminator sequencing using the single-dye method remains inefficient. We have successfully established a new sequencing system using a thermostable DNA polymerase and a thermal cycling approach, combined with the use of four fluorescent...

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Bibliographic Details
Published in:Analytical Sciences 1997/10/10, Vol.13(5), pp.735-739
Main Authors: TAKUMI, Toru, FUJIWAKE, Hideshi, KURACH, Yoshihisa
Format: Article
Language:English
Subjects:
Biological and medical sciences
Biotechnology
Dye terminator
fluorescence
Fundamental and applied biological sciences. Psychology
Genetic engineering
Genetic technics
Methods. Procedures. Technologies
sequencer
Synthetic digonucleotides and genes. Sequencing
thermal cycle
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Summary:Automated DNA sequencers have been developed rapidly, but dye terminator sequencing using the single-dye method remains inefficient. We have successfully established a new sequencing system using a thermostable DNA polymerase and a thermal cycling approach, combined with the use of four fluorescent dideoxynucleosides on an automated DNA sequencer. Using double-strand DNAs as templates and synthesized oligonucleotides as primers, this method gave 550-900bp read lengths (with a mean read length of 755bp). This dye terminator method is both reliable and economical and should become a general technique for walking sequencing using automated DNA sequencers.
ISSN:0910-6340
1348-2246
DOI:10.2116/analsci.13.735