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PMID: 2191292 Published · ppublish English Journal Article Research Support, U.S. Gov't, Non-P.H.S.

Imaging of DNA sequences with chemiluminescence.

Proceedings of the National Academy of Sciences of the United States of America ·Vol. 87 ·No. 12 ·1990-06-00 ·Pages 4514-8

Tizard R, Cate RL, Ramachandran KL, Wysk M, Voyta JC, Murphy OJ, Bronstein I

Abstract

We have coupled a chemiluminescent detection method that uses an alkaline phosphatase label to the genomic DNA sequencing protocol of Church and Gilbert [Church, G. M. & Gilbert, W. (1984) Proc. Natl. Acad. Sci. USA 81, 1991-1995]. Images of sequence ladders are obtained on x-ray film with exposure times of less than 30 min, as compared to 40 h required for a similar exposure with a 32P-labeled oligomer. Chemically cleaved DNA from a sequencing gel is transferred to a nylon membrane, and specific sequence ladders are selected by hybridization to DNA oligonucleotides labeled with alkaline phosphatase or with biotin, leading directly or indirectly to deposition of enzyme. If a biotinylated probe is used, an incubation with avidin-alkaline phosphatase conjugate follows. The membrane is soaked in the chemiluminescent substrate (AMPPD) and is exposed to film. Dephosphorylation of AMPPD leads in a two-step pathway to a highly localized emission of visible light. The demonstrated shorter exposure times may improve the efficiency of a serial reprobing strategy such as the multiplex sequencing approach of Church and Kieffer-Higgins [Church, G. M. & Kieffer-Higgins, S. (1988) Science 240, 185-188].

MeSH Terms

Adamantane/analogs & derivatives Adenosine Triphosphate Alkaline Phosphatase Base Sequence DNA/genetics Genetic Techniques Luminescent Measurements Molecular Sequence Data Nucleic Acid Hybridization Oligonucleotide Probes Phosphorus Radioisotopes Plasmids

Chemicals

Oligonucleotide Probes Phosphorus Radioisotopes 3-(2'-spiroadamantane)-4-methoxy-4-(3''-phosphoryloxy)phenyl-1,2-dioxetane Adenosine Triphosphate DNA Alkaline Phosphatase Adamantane

Authors & Affiliations

7 authors, click to expand affiliations / ORCID

Tizard R

Biogen, Inc., 14 Cambridge Center, MA 02142.

Cate R L

Ramachandran K L

Wysk M

Voyta J C

Murphy O J

Bronstein I

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Article Info

Journal

Proceedings of the National Academy of Sciences of the United States of America

Abbr.

Proc Natl Acad Sci U S A

ISSN

0027-8424

DOI

10.1073/pnas.87.12.4514

Published

1990-06-00

Pages

4514-8

Language

English

Region

United States

NLM ID

7505876

PMCID

PMC54146

Subset

External Links

PubMed source DOI: 10.1073/pnas.87.12.4514 PMC Full Text View journal details

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