Nonradioactive labeling and high-sensitive detection of PCR products.
The polymerase chain reaction (PCR) represents the most common and widespread method for the direct amplification of specific sequences of nucleic acid target molecules. Incorporation of nonradioactive labeled nucleotides during PCR by Taq DNA polymerase results in directly detectable amplification products or generates nonradioactively labeled probes for nucleic acid hybridization. Here we provide a reliable and easy to follow protocol for direct incorporation of digoxigenin-(DIG) or biotin-labeled nucleotides during PCR. The combination of high-efficient PCR amplification and high-sensitive digoxigenin technology is leading to the detection of single DNA molecules by applying digoxigenin-specific antibodies in an ELISA-type detection reaction. Following a transfer to nylon membranes, the detection of digoxigenin-labeled amplification products can also be accomplished either with a colorimetric or a chemiluminescent reaction. Using the digoxigenin-labeled amplification products as hybridization probes, sensitivities in the 0.1-pg range are obtained in Southern blot procedures.
Reischl
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U
Institute for Medical Microbiology and Hygiene, University of Regensburg, Germany.
Rüger
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R
Kessler
C
C
D001709:Biotechnology; D001710:Biotin; D015139:Blotting, Southern / Q000379:methods; D004247:DNA / Q000235:genetics; D004259:DNA-Directed DNA Polymerase; D004076:Digoxigenin; D004587:Electrophoresis, Agar Gel / Q000379:methods; D005784:Gene Amplification; D007118:Immunoassay / Q000379:methods; D015335:Molecular Probes / Q000302:isolation & purification; D009693:Nucleic Acid Hybridization; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000706:statistics & numerical data; D012313:RNA / Q000235:genetics; D012680:Sensitivity and Specificity; D019914:Taq Polymerase
D015335:Molecular Probes; D012313:RNA; D001710:Biotin; D004247:DNA; D019914:Taq Polymerase; D004259:DNA-Directed DNA Polymerase; D004076:Digoxigenin