Direct in-gel hybridization of DNA with digoxigenin-labeled probes.
In-gel hybridization with digoxigenin (DIG)-labeled probes has been shown to detect complementary DNA sequences in dried agarose gels. Gels dried at room temperature or at 55 degrees C in an oven do not show detectable changes in the sensitivity of detection. However, gels dried under vacuum seem to lose the sensitivity by approx 8- to 10-fold. In-gel hybridization after blotting high molecular weight T7 DNA (40 kbp) onto nylon membranes has been demonstrated to transfer the DNA to the membrane inefficiently. In-gel hybridization of DIG-labeled probes with the complementary DNA sequences has been determined to detect as little as 0.05 ng of 40-kbp T7 DNA and single copies of the erythromycin resistance marker gene ermA. Nonradioactive in-gel hybridization provides better quantitation of nucleic acids than filter hybridization in Southern and Northern blot analysis.
Khan
Saeed A
SA
Division of Microbiology, National Center for Toxicological Research, US Food and Drug Administration, Jefferson, AR, USA.
Nawaz
Mohamed S
MS
D001426:Bacterial Proteins / Q000235:genetics; D017123:Bacteriophage T7 / Q000235:genetics; D001483:Base Sequence; D004247:DNA / Q000235:genetics* / Q000302:isolation & purification*; D017931:DNA Primers / Q000235:genetics; D004279:DNA, Viral / Q000235:genetics / Q000302:isolation & purification; D004076:Digoxigenin; D005782:Gels; D018628:Gene Dosage; D005798:Genes, Bacterial; D008780:Methyltransferases / Q000235:genetics; D015336:Molecular Probe Techniques; D009693:Nucleic Acid Hybridization / Q000379:methods*; D015345:Oligonucleotide Probes / Q000235:genetics*; D012492:Salts; D013211:Staphylococcus aureus / Q000235:genetics; D014618:Vacuum
D001426:Bacterial Proteins; D017931:DNA Primers; D004279:DNA, Viral; D005782:Gels; D015345:Oligonucleotide Probes; D012492:Salts; D004247:DNA; D008780:Methyltransferases; C431709:ErmA protein, Bacteria; D004076:Digoxigenin