Description
General description
Kit for radioactive or nonradioactive labeling of DNA by the nick translation method. This method utilizes a combination of DNase and DNA Polymerase to nick one strand of the DNA helix, then incorporates labeled nucleotides as the polymerase examines, or "proofreads" the nicked site.
Application
The kit is used for labeling of DNA with radioactive or modified dNTPs. Probes labeled by nick translation are used in many different hybridization techniques. These include use as probes in:
Screening gene banks by colony- or plaque hybridization
DNA or RNA transfer hybridizations
In situ hybridization
Reassociation kinetic studies
Nick translation kit has been used in FISH (fluorescence in situ hybridization) based labeling of tick and bacterial artificial chromosome. It has been used in labeling the probe (genomic DNA) in order to obtain GISH (genomic in situ hybridization) signals.
Biochem/physiol Actions
Heat inactivation: Stop the reaction by adding 1μl 0.5 M EDTA (pH 8.0) and/or by heating to 65 °C for 10 minutes.
The nick translation method is based on the ability of DNase I to introduce randomly distributed nicks into DNA at low enzyme concentrations in the presence of Mg²⁺. E. coli DNA polymerase I synthesizes DNA complementary to the intact strand in a 5′→3′ direction using the 3′-OH termini of the nick as a primer. The 5′→3′ exonucleolytic activity of DNA polymerase I simultaneously removes nucleotides in the direction of synthesis. The polymerase activity sequentially replaces the removed nucleotides with isotope-labeled or hapten-labeled deoxyribonucleoside triphosphates. At low temperature (+15°C), the unlabeled DNA in the reaction is thus replaced by newly synthesized labeled DNA.
Features and Benefits
Assay Time: 45minutes
Specific Activity: The level of specific labeling and the incorporation rate are dependent on the ratio of substrate DNA to labeled deoxyribonucleoside triphosphate, e.g., the kinetics and labeling levels obtained are identical in assays containing 0.1μg DNA and 20μCi dXTP or 0.5μg DNA and 100μCi dXTP.
The standard assay will give a specific activity of 3 x 10⁸dpm/μg, corresponding to 65% incorporation with different substrate DNAs (e.g., pBR322, λDNA, DNA fragments) in 35minutes.
Sample Materials
Supercoiled and linearized plasmid DNA
Supercoiled and linearized cosmid DNA
BAC DNA or human genomic DNA
Purified PCR products
Note: Denaturing of the template before nick translation is not required.
Packaging
1 kit containing 7 components.
Preparation Note
Working concentration: The recommended working concentration with T7, T3 or SP6 RNA Polymerases is 1 mM.
Working solution: DNA
The DNA must be in low-salt concentration solution.
dATP, dGTP, dTTP
If the same labeled deoxyribonucleoside triphosphate is used repeatedly, we recommend the preparation of a mixture of equal parts of the other three triphosphates for convenience.
Prepare the dATP, dGTP, dTTP mixture by making a 1:1:1 mixture of solution 2, solution 4, and solution 5.
Other Notes
For life science research only. Not for use in diagnostic procedures.
