Uses
For generation of highly sensitive probes for
in situ hybridization labeled with Biotin-16-dUTP.
Note: The mix can also be used for filter hybridization techniques, however, for highly sensitive filter hybridization probes, we recommend that you use Biotin-High Prime from Roche Applied Science.
For nonradioactive labeling of
in situ probes with other haptens, Roche Applied Science offers the DIG-Nick Translation Mix or the Nick Translation Mix.
General Description
Convenient enzyme and nucleotide mixture.
Nick translation 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. The molar ratio of Biotin-16-dUTP to dTTP is adjusted to ensure that every 20
th to 25
th nucleotide in the newly synthesized DNA is modified with biotin. This density of haptens in the DNA yields the highest sensitivity in the immunological detection reaction.
Large plasmids, cosmids, and PCR fragments are all regularly used with this method, and the DNA can be linearized or supercoiled. Individual templates produce consistent results in the standard 90-minutes reaction, and result in an average probe length of 200 base pairs up to 500 base pairs.
