The MegaPrime™ DNA Labelling System is a simple kit to radioactively label DNA or cDNA sequences for use in nucleic acid hybridizations. Since the discovery of the use of random sequence oligonucleotides to initiate DNA synthesis on multiple sites of denatured template DNA, this approach has been widely used for labeling DNA probes. The primer-template complex is a substrate for the ‘Klenow” fragment of the DNA Polymerase I. The latter can incorporate labeled nucleotides into the newly synthesized DNA. This represents a relatively simple method to build probes in a home-made fashion for blot-based applications.
The MegaPrime™ DNA Labelling System consists of a set of solution reagents and enzyme that can be easily stored at –20ºC. The main feature of the MegaPrime™ DNA Labelling System resides in the use of nonamer primers (hence the name). These primers are longer than the hexanucleotides which were used when the technique was established. Nonamers allow for more efficient priming from the template DNA, resulting in faster and more efficient DNA labeling than in the past. The system allows double-strand DNA to be labeled in vitro to high specific activity 32P (or other radionuclides). The incubation time required at 37ºC is reduced to 10 min. To label cDNA probes we normally use 32P at 3000 mCi/mmol, and purified DNA concentrated in the range of ng to ug/ul.
We routinely use the MegaPrime™ DNA Labelling System to radiolabel cDNA probes for Northern blot hybridizations. From the standard conditions specified above, we normally obtain 1-2 x 107 cpm of radiolabeled probe which can be used for two or more blots. We particularly appreciate the stability of the kit components (including the enzyme), which are stored at –20 ºC: In the last few years they have been subjected to several heat shocks without apparently undergoing any degradation.
I am fully satisfied with the performance of the MegaPrime™ DNA Labelling System over the last few years. Therefore, it will remain my system for DNA labeling.
Research Associate
Department of Histology and Medical Embryology
University of Rome, La Sapienza