A two-step hybridization method for chemiluminescent detection of single copy genes.
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.
Nguyen
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Bio-Rad Laboratories, Richmond, CA 94806.
Witney
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Tumolo
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D000199:Actins / Q000235:genetics; D000218:Adamantane / Q000031:analogs & derivatives; D015139:Blotting, Southern; D004247:DNA / Q000032:analysis*; D015342:DNA Probes; D004563:Electrochemistry; D005169:Factor VIII / Q000235:genetics; D016259:Genes, myc; D005821:Genetic Techniques; D015894:Genome, Human; D006801:Humans; D008163:Luminescent Measurements; D008567:Membranes, Artificial; D009693:Nucleic Acid Hybridization
D000199:Actins; D015342:DNA Probes; D008567:Membranes, Artificial; C061523:3-(2'-spiroadamantane)-4-methoxy-4-(3''-phosphoryloxy)phenyl-1,2-dioxetane; D005169:Factor VIII; D004247:DNA; D000218:Adamantane