Rapid and reliable fluorescent cycle sequencing of double-stranded templates

McCombie, W. R., Heiner, C., Kelley, J. M., Fitzgerald, M. G., Gocayne, J. D. (1992) Rapid and reliable fluorescent cycle sequencing of double-stranded templates. DNA sequence : the journal of DNA sequencing and mapping, 2 (5). pp. 289-296. ISSN 10425179 (ISSN)

URL: http://www.ncbi.nlm.nih.gov/pubmed/1633326

Abstract

Automated DNA sequencing is an extremely valuable technique which requires very high quality DNA templates to be carried out successfully. While it has been possible to readily produce large numbers of such templates from M13 or other single-stranded vectors for several years, the sequencing of double-stranded DNA templates using the ABI 373 DNA Sequencer has had a considerably lower success rate. We describe how the combination of a new fluorescent, dideoxy sequencing method, called cycle-sequencing, coupled with modifications to template isolation procedures based on Qiagen columns, makes fluorescent sequencing of double-stranded templates a reliable procedure. From a single five milliliter culture enough DNA can be isolated (up to 20 micrograms) to do 4-8 sequencing reactions, each of which yields 400-500 bases of high quality sequence data. These procedures make the routine use of double-stranded DNA templates a viable strategy in automated DNA sequencing projects.

Item Type: Paper
Uncontrolled Keywords: DNA DNA, Single Stranded single stranded DNA article chemistry DNA template fluorescence isolation and purification molecular cloning molecular genetics nucleotide sequence plasmid Base Sequence Cloning, Molecular DNA, Single-Stranded Molecular Sequence Data Plasmids Templates, Genetic
Subjects: bioinformatics > genomics and proteomics > genetics & nucleic acid processing > DNA, RNA structure, function, modification
bioinformatics > genomics and proteomics > genetics & nucleic acid processing
Investigative techniques and equipment
Investigative techniques and equipment > assays
Investigative techniques and equipment > cloning
Investigative techniques and equipment > assays > cloning
CSHL Authors:
Communities: CSHL labs > McCombie lab
Depositing User: Matt Covey
Date Deposited: 23 Apr 2013 16:43
Last Modified: 23 Apr 2013 16:43
URI: http://repository.cshl.edu/id/eprint/28238

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