Radioactivity is a vital tool for molecular biology studies because of the sensitivity it offers to an experimental system. Nucleic acid hybridization provides a means for detecting DNA or RNA sequences that are complementary to any desired nucleic acid. The radioactive oligonucleotide is used as a probe for hybridization to the complementary DNA or RNA sequences that are to be detected. Various methods for generating radiolabeled DNA probes: polymerase chain reaction (PCR), nick translation, end labeling and random primer labeling, have been developed over the time.
The NEBlot® Kit is a convenient method of labeling DNA. It is based on the principle of random primer labeling. Random octamers act as primers for the Klenow enzyme to synthesize the complementary strand of denatured linear DNA in the presence of dNTPs. The kit is compatible with any radiolabeled dNTP. The labeling reaction quickly (30 minutes) generates high specific activity probes of >1x109 dpm/µg, from as little as 25 ng to 2 µg of DNA. The reaction is efficient and consistent. Although I have not tried it, NEBlot® can also label DNA in low melting point (LMP) agarose, thus overcoming the limitations of the older nick translation methodology. The NEBlot® Kit is faster than a PCR labeling method, where DNA is amplified in the presence of labeled dNTPs. However, very little DNA (10-100 pg) can be labeled using PCR, with extensive optimization. With the aid of the NEBlot® Kit, 100 ng of DNA can be labeled with 75% incorporation in 15 minutes. Also, ~5 ng DNA can be labeled with up to 50% incorporation if the reaction is incubated for 2 hours.
The kit is stored at -20ºC and includes random primers in 10X labeling buffer, E. coli DNA polymerase Klenow fragment (3’→5’ exo‾), all 4 dNTPs (unlabeled) and nuclease-free water. Control DNA is also provided with the kit to assess the kit’s performance. For setting up the labeling, reaction buffer, dNTPs (including radiolabeled dNTP which is not provided with the kit) and Klenow fragment are combined and incubated at 37ºC for 1 hour.
Label incorporation occurs in 30 minutes; however, it is better to incubate for 1 hour to achieve maximum activity. After synthesis, gel filtration or spin columns can be used to purify labeled probe. We use Sephadex® G-50 columns (from GE Healthcare) for probe purification. Similar products from other companies claim that the labeled probe generated by their kit is ready for hybridization without purification. I would always prefer to purify my probe, as it will help in reducing nonspecific background (because of unincorporated labeled dNTPs). The probes generated by the NEBlot® Kit are suitable for applications such as probing of Southern and Northern blots, screening gene libraries and in situ hybridization.
We are studying the chromatin organization of promoters of cell cycle regulatory genes. The native chromatin is digested with non-specific endonucleases and the desired regions are analyzed by Southern hybridization. The probes are generated either by PCR or restriction digestion of cloned DNA. We have used the NEBlot® Kit for 4 years to generate DNA probes for hybridization experiments; we have used this kit to label DNA ranging from 400 bp to 1 kb. I have always been satisfied with the performance of the kit. Because caution has to be taken while handling radioactive material, it is worth noting that NEB has chemiluminescent detection kits. These kits generate probes that are stable for extended periods of time, unlike 32P-labeled probes. However, we are comfortable with radioactivity as it is used for numerous experiments in our laboratory. Also, chemiluminescent detection kits are comparatively expensive.
Apart from the simplicity and efficiency of the system, the NEBlot® Kit is economical. In comparison to random primer labeling kits offered by other companies, the price is relatively low. Last but not the least, the kit is guaranteed for 1 year. This guarantee is not an issue, however, since the product does not give any problems. But this guarantee adds to the user’s confidence in the reliability of the product.
The NEBlot® Kit would undoubtedly be graded “excellent” in my view.
Nidhi Vishnoi
Senior Research Fellow
Cancer Research Institute, Advanced Centre for Treatment, Research and Education in Cancer (ACTREC), TMC.
Chemical Carcinogenesis