Imaging of DNA sequences with chemiluminescence
| Autor: | Mark Wysk, John C. Voyta, Owen J. Murphy, Richard L. Cate, Richard Tizard, Kuzhalmannam L. Ramachandran, Irena Y. Bronstein |
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| Rok vydání: | 1990 |
| Předmět: |
Molecular Sequence Data
Phosphatase Adamantane DNA sequencing chemistry.chemical_compound Nucleic acid thermodynamics Adenosine Triphosphate Multidisciplinary Base Sequence Oligonucleotide DNA–DNA hybridization Nucleic Acid Hybridization DNA Alkaline Phosphatase Molecular biology Genetic Techniques chemistry Biochemistry Biotinylation Luminescent Measurements Alkaline phosphatase Oligonucleotide Probes Phosphorus Radioisotopes Plasmids Research Article |
| Zdroj: | Proceedings of the National Academy of Sciences. 87:4514-4518 |
| ISSN: | 1091-6490 0027-8424 |
| DOI: | 10.1073/pnas.87.12.4514 |
| Popis: | 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]. |
| Databáze: | OpenAIRE |
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