For optimized labeling of differential display fragments with radioactively labeled dATP.
The HotPrime DNA labeling kit features three unique improvements over the traditional
"random priming" method which allow for radioactive labeling of DNA probes with at least 5-10X more specific radioactivity. It is designed to
efficiently label cDNA probes isolated from differential display for Northern or Southern blot analysis as well as library screening. However,
the HotPrime Kit can also label DNA probes for any other application to a similar high specific activity.
The first improvement over traditional random-prime kits involves using random decamers instead of hexamers. This ensures more efficient priming
of DNA probes of any sequence based on the observation that primers shorter than 9 bases are inefficient in initiating DNA synthesis (Williams et
al., 1991, Nucleic Acids Res., 18:6531-6535).
Second, because the cDNA fragments from differential display are relatively short (150-700 bp), the incorporation of anchored oligo-dT primers
into the labeling buffer ensures the "full length" anti-sense cDNA probes to be labeled, which greatly increases the chance for signal detection
on the Northern blot or library screening.
Third, the use of radioactive dATP instead of dCTP allows labeling of DNA to the highest specific activity by taking the advantage of the AT rich
nature of the 3' untranslated regions of mRNA detected by differential display.
The HotPrime DNA Labeling Kit is also available with dNTP (-dCTP) for use with a-[32P] dCTP (Cat. # H501-C).
| ||Cat. No.|
|HotPrime DNA Labeling Kit||H501
Northern blot analysis of differential expression of cDNA probe isolated by
The band of interest from differential display was excised (A, arrowhead),reamplified and labeled with the Hotprime DNA labeling kit as a probe for Northern
blot confirmation (B).