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Direct sequencing of lambda DNA from crude lysates using an improved linear amplification technique

Lasham, Annette; Darlison, Mark G.

Authors

Annette Lasham

Mark G. Darlison



Abstract

We described an improved method for directly sequencing lambda (λ) DNA that has been isolated from either crude cleared lysates or plate lysates. This protocol does not require that the DNA be obtained from bacteriophage particles that have been purified by caesium chloride centrifugation. Nanogram quantities of λ DNA are unidirectionally amplified using a radioactively-labelled oligonucleotide primer, and Thermus aquaticus (Taq) DNA polymerase, in the presence of T4 gene 32 protein (gp 32). The amplification/sequencing reactions are then incubated with terminal deoxynucleotidyl transferase (TdT) and all four deoxynucleotide triphosphates to elongate any prematurely-arrested products. This procedure, which is a modification of a previously-published method, results in a significant improvement in the quality and amount of DNA sequence information that can be obtained from λ templates. Although it was developed to sequence DNA directly from λEMBL3 recombinants, it can also be used with cosmid DNA, M13 and plasmid DNA, and polymerase chain reaction (PCR) amplification products, yielding excellent ladders in each case. In addition, our method resolves the nucleotide sequences of double-stranded plasmid templates that are difficult to determine by conventional dideoxynucleotide sequencing protocols because of 'stalling', in which bands appear at the same position in all four lanes.

Citation

Lasham, A., & Darlison, M. G. (1993). Direct sequencing of lambda DNA from crude lysates using an improved linear amplification technique. Molecular and Cellular Probes, 7(1), 67-73. doi:10.1006/mcpr.1993.1009

Journal Article Type Article
Acceptance Date Feb 1, 1993
Publication Date 1993-02
Deposit Date Aug 5, 2016
Journal Molecular and Cellular Probes
Print ISSN 0890-8508
Electronic ISSN 0890-8508
Publisher Elsevier
Peer Reviewed Peer Reviewed
Volume 7
Issue 1
Pages 67-73
DOI https://doi.org/10.1006/mcpr.1993.1009
Keywords Cell Biology; Molecular Biology
Public URL http://researchrepository.napier.ac.uk/Output/328055






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