Imaging of DNA sequences with chemiluminescence
- Biogen, Inc., Cambridge, MA (United States)
- Tropix, Inc., Bedford, MA (United States)
We have coupled a chemiluminescent method for detecting oligonucleotides labeled with alkaline phosphatase to the genomic DNA sequencing protocol of Church and Gilbert. Images of sequence ladders obtained on x-ray film in a 30 minute exposure are comparable to those from a 40 hour exposure with 3000 Ci/mmol {sup 32}P probes. Chemically cleaved DNA from a sequencing gel is transferred to a nylon membrane, and specific sequence ladders are selected by hybridization to an oligonucleotide probe conjugated either to biotin or to alkaline phosphates. If biotinylated probe is used, then an avidin-alkaline phosphatase conjugate is subsequently bound. This membrane, bearing immobilized alkaline phosphatase, is incubated with the commercially available chemiluminescent substrate disodium 3-(4-methoxyspiro[1,2-dioxetone-3,2{prime}-tricyclo[3.3.1.1.{sup 3.7}]decan]-4-yl)phenyl phosphate. (AMPPD) Dephosphorylation of AMPPD leads in a two step pathway to a highly localized emission of visible light.
- Research Organization:
- Tropix, Inc., Bedford, MA (United States)
- Sponsoring Organization:
- USDOE, Washington, DC (United States)
- DOE Contract Number:
- AC05-89ER80752
- OSTI ID:
- 10141935
- Report Number(s):
- CONF-8910482-1; ON: DE92011906
- Resource Relation:
- Conference: Human genome I: international conference on the status and future of research on the human genome,San Diego, CA (United States),2-4 Oct 1989; Other Information: PBD: [1989]
- Country of Publication:
- United States
- Language:
- English
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