A two-step hybridization method for chemiluminescent detection of single copy genes
- PMID: 1503762
A two-step hybridization method for chemiluminescent detection of single copy genes
Abstract
We have developed a technique for the chemiluminescent detection of single copy genes that eliminates the high backgrounds and problems with probe labeling associated with existing methods. The procedure employs a primary hybridization of single-stranded probe DNA to immobilized target DNA, a secondary hybridization with a covalently cross-linked oligonucleotide-alkaline phosphatase conjugate, followed by incubation in the chemiluminescent substrate AMPPD and detection on x-ray film. The key to the success of this method is that the primary probe contains a region complementary to the target DNA as well as to the oligonucleotide sequence of the secondary probe-alkaline phosphatase conjugate. Here we report our results using the two-step hybridization procedure to detect single copy genes from genomic Southern blots.
Similar articles
-
Sensitive chemiluminescent detection of digoxigenin-labeled nucleic acids: a fast and simple protocol and its applications.Biotechniques. 1992 Jan;12(1):104-13. Biotechniques. 1992. PMID: 1370901
-
Rapid and sensitive detection of DNA in Southern blots with chemiluminescence.Biotechniques. 1990 Mar;8(3):310-4. Biotechniques. 1990. PMID: 2331371
-
Genomic Southern analysis with alkaline-phosphatase-conjugated oligonucleotide probes and the chemiluminescent substrate AMPPD.Genet Anal Tech Appl. 1991 May;8(3):102-6. doi: 10.1016/1050-3862(91)90044-r. Genet Anal Tech Appl. 1991. PMID: 1905941
-
Nucleic acid detection using non-radioactive labelling methods.Mol Cell Probes. 1995 Jun;9(3):145-56. doi: 10.1006/mcpr.1995.0023. Mol Cell Probes. 1995. PMID: 7477006 Review.
-
Hybridization analysis of DNA blots.Curr Protoc Immunol. 2001 May;Chapter 10:Unit 10.6B. doi: 10.1002/0471142735.im1006bs06. Curr Protoc Immunol. 2001. PMID: 18432698 Review.
MeSH terms
Substances
LinkOut - more resources
Other Literature Sources
Molecular Biology Databases