Evaluation of nonisotopic labeling and detection techniques for nucleic acid hybridization.
We have used a double-stranded DNA probe linked to the cystic fibrosis locus to detect a single-copy gene from varying amounts of genomic DNA, with Southern blot analysis. The DNA plasmid probe was labeled with either biotin or digoxigenin. Biotin or digoxigenin was then linked to alkaline phosphatase (ALP) with use of streptavidin or anti-digoxigenin antibodies, respectively. ALP activity was then detected with a chromogenic (BCIP/NBT), chemiluminogenic (AMPPD), or fluorogenic (HNPP) substrate. Our results suggest that biotin and digoxigenin perform similarly and that the three substrates exhibit similar detectability under appropriate substrate incubation times: 20-120 min (AMPPD), 2-12 h (HNPP), and 18-48 h (BCIP/NBT). Under optimised conditions and the probe used, these methods detect single-copy genes from as little as 0.3 micrograms of total genomic DNA.
Department of Clinical Biochemistry, Toronto Hospital, Ontario, Canada.
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Diamandis
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Hassapoglidou
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Bean
D001710:Biotin; D015139:Blotting, Southern; D004247:DNA / Q000235:genetics / Q000302:isolation & purification; D015342:DNA Probes; D004076:Digoxigenin; D005069:Evaluation Studies as Topic; D006801:Humans; D009693:Nucleic Acid Hybridization / Q000379:methods*
D015342:DNA Probes; D001710:Biotin; D004247:DNA; D004076:Digoxigenin