Biotin 3’ End DNA Labeling Kit From Pierce Biotechnology

The Biotin 3’ End DNA Labeling Kit from Pierce Biotechnology incorporates 1-3 biotinylated ribonucleotides onto the 3’ end of DNA strands by using Terminal Deoxynucleotidyl Transferase (TdT). The TdT prefers to label single-stranded DNA, but can also label double-stranded DNA with blunt or 3’ overhangs, but at a lower efficiency. This procedure is ideal for non-isotopic EMSA applications as well as Northern or Southern blot hybridizations. Pierce offers kits to visualize the biotin labeled DNA, including the LightShift Chemiluminescent EMSA Kit.

The materials needed, but not provided for biotin labeling include ultrapure water, 1 micromolar of the oligo to be labeled, a 37ºC heat block or water bath, 0.2 M EDTA (pH 8.0), chloroform:isoamyl alcohol (24:1), a microcentrifuge and tubes. The protocol is straightforward and easy.

For techniques that require double-stranded DNA, like EMSAs, Pierce suggests to biotin label the complementary oligos separately and then anneal the two strands together. They provide the procedure to do this in the protocol. I followed their recommendation with successful results, so I do not know what the labeling efficiency is like if you try to label double-stranded DNA.

Once you have added the required volumes of reagents into the labeling reaction, it is incubated for 30 minutes at 37ºC. The reaction is then stopped with the EDTA and the TdT is extracted with the chloroform:isoamyl alcohol. After you remove and save the aqueous phase, containing the labeled oligo of interest, you can determine the labeling efficiency by following the protocol on the next page of the instruction manual. If you do not need to know the exact concentrations of your labeled oligo, there is no need to determine labeling efficiency with this protocol. Instead, you can load the labeled oligo at various volumes onto a gel and use the LightShift EMSA Kit in order to visualize the oligo and determine the best intensity and dilution for your application.

If determining the concentration of the labeled oligo is necessary, two options are provided in the protocol for estimating the biotin labeling efficiency. Option one utilizes a dot/slot blotting apparatus. The materials required but not supplied for this protocol are: positively charged nylon membrane, dot blotting apparatus, TE buffer, 96-well microplate, multi-channel pipette, microcentrifuge and tubes, UV lamp or cross-linking device equipped with 254 nm bulbs or 312 nm transilluminator, streptavidin-HRP and chemiluminescent substrate (e.g. LightShift substrate), and x-ray film or CCD camera. The protocol is detailed as to what volumes of each reagent to use. A chart is provided for making standards with various percentages of biotin and control oligos (provided in the kit). The protocol also describes how to prepare the biotin-labeled sample oligo and how to load all the samples on the dot/slot blot apparatus with several dilutions of each loaded onto the system. Since all dot blot systems are different, you will need to be familiar with how to operate the one you own. The system we use in our lab is the Bio-Dot Microfiltration system from Bio-Rad. This system works nicely and is easy to operate. After blotting of the samples is complete, the membrane is cross-linked. The protocol offers three different options to do this. We chose the third option where we put the membrane face down on the UV transilluminator for 15 min. After cross-linking, the LightShift chemiluminescent EMSA Kit from Pierce was used to detect the biotin-labeled oligos. Once the biotin is detected either using X-ray film or a CCD camera, the picture was scanned and the bands were quantified using Quantity One software from Bio-Rad. Using the values obtained from quantification, the percent biotin of the sample oligo can be determined by comparing it to the standard curve produced with the standards. Option two for determining the labeling efficiency is a modification to the one described above, but uses smaller total volumes and no dot/slot blot apparatus is required. Instead, the dot blot is created by hand spotting the samples. After that is complete, the cross-linking, detection, and analysis would be performed the same as above.

This kit has been useful in my laboratory in studying aptamer-protein binding as well as for performing NFkB and AP-1 EMSAs. The kit states that the unlabeled control oligo that is provided should be labeled with 70% or greater efficiency. In my hands, I have achieved over 80% labeling efficiency with this control oligo and 65% to 75% labeling efficiency with my sample oligo. I would suggest any laboratory interested in a non-isotopic procedure to study DNA binding purchase this kit.