Imaging of DNA sequences with chemiluminescence

Journal Article · Fri Jun 01 00:00:00 EDT 1990 · Proceedings of the National Academy of Sciences of the United States of America; (USA)

DOI:https://doi.org/10.1073/pnas.87.12.4514· OSTI ID:6718806

Tizard, R; Cate, R L; Ramachandran, K L; Wysk, M; Voyta, J C; Murphy, O J; Bronstein, I ^[1]

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1. Biogen, Inc., 14 Cambridge Center, MA (USA)

We have coupled a chemiluminescent detection method that uses an alkaline phosphatase label to the genomic DNA sequencing protocol of Church and Gilbert . 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.

• OSTI ID: 6718806
• Journal Information: Proceedings of the National Academy of Sciences of the United States of America; (USA), Journal Name: Proceedings of the National Academy of Sciences of the United States of America; (USA) Vol. 87:12; ISSN PNASA; ISSN 0027-8424
• Country of Publication: United States
• Language: English

Related Subjects

550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
ALKALINE PHOSPHATASE
ATP
BETA DECAY RADIOISOTOPES
BETA-MINUS DECAY RADIOISOTOPES
BIOLOGICAL PATHWAYS
CELL CONSTITUENTS
CHEMILUMINESCENCE
DAYS LIVING RADIOISOTOPES
DNA HYBRIDIZATION
DNA SEQUENCING
ELECTROMAGNETIC RADIATION
ENZYMES
ESTERASES
HYBRIDIZATION
HYDROLASES
IONIZING RADIATIONS
ISOTOPE APPLICATIONS
ISOTOPES
LIGHT NUCLEI
LUMINESCENCE
NUCLEI
NUCLEIC ACIDS
NUCLEOTIDES
ODD-ODD NUCLEI
OLIGONUCLEOTIDES
ORGANIC COMPOUNDS
PHOSPHATASES
PHOSPHORUS 32
PHOSPHORUS ISOTOPES
PLASMIDS
RADIATIONS
RADIOISOTOPES
STRUCTURAL CHEMICAL ANALYSIS
TRACER TECHNIQUES
X RADIATION