Nonradioactive labeling and high-sensitive detection of PCR products
- 1 June 1994
- journal article
- research article
- Published by Springer Nature in Molecular Biotechnology
- Vol. 1 (3) , 229-240
- https://doi.org/10.1007/bf02921691
Abstract
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 nonradioactivc labeled nucleotides during PCR byTaq 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, sensilivities in the 0.1-pg range are obtained in Southern blot procedures.Keywords
This publication has 19 references indexed in Scilit:
- The digoxigenin:anti-digoxigenin (DIG) technology—a survey on the concept and realization of a novel bioanalytical indicator systemMolecular and Cellular Probes, 1991
- Non-radioactive Labeling and Detection of Nucleic Acids. III Applications of the Digoxigenin SystemBiological Chemistry Hoppe-Seyler, 1990
- Non-radioactive Labeling and Detection of Nucleic Acids. II. Optimization of the Digoxigenin SystemBiological Chemistry Hoppe-Seyler, 1990
- Non-radioactive Labeling and Detection of Nucleic Acids. I. A Novel DNA Labeling and Detection System Based on Digoxigenin: Anti-Digoxigenin ELISA Principle (Digoxigenin System)Biological Chemistry Hoppe-Seyler, 1990
- Non-radioactive Labeling and Detection of Nucleic Acids. IV. Synthesis and Properties of Digoxigenin-modified 2´ -Deoxyuridine-5’ -triphosphates and a Photoactivatable Analog of Digoxigenin (Photodigoxigenin)Biological Chemistry Hoppe-Seyler, 1990
- 1,2‐Dioxetanes: Novel chemiluminescent enzyme substrates. Applications to immunoassaysJournal of Bioluminescence and Chemiluminescence, 1989
- The ligation amplification reaction (LAR)—Amplification of specific DNA sequences using sequential rounds of template-dependent ligationGenomics, 1989
- Exponential Amplification of Recombinant- RNA Hybridization ProbesBio/Technology, 1988
- Primer-Directed Enzymatic Amplification of DNA with a Thermostable DNA PolymeraseScience, 1988
- Enzymatic Amplification of β-Globin Genomic Sequences and Restriction Site Analysis for Diagnosis of Sickle Cell AnemiaScience, 1985