Matthew Freeman

OLIGO-LABELLED DNA PROBES

This technique is an alternative to nick translation, and is generally superior. I find it less fussy, and it routinely gives considerably hotter probes. It also has the advantage that even quite dirty DNA preps work fine, and it is possible to label fragments in LGT agarose ;with no purification.
The 5X oligo-labelling buffer ;(OLB) can be made in large batches and stored at -20° for at least a year. If you do this, keep it in aliquots of a size such that you thaw and refreeze a particular tube for not more than a month or two - after this it may become less effective.oligo-labelled DNA probes;
For all normal uses it is only necessary to use one hot nucleotide (I use 32P-dATP). The OLB has the necessary cold nucleotides, so you need to make another one if you want to use two hot ones.

The OLB is made from several component solutions.

Solution O: 1.25M Tris HCl, pH8.0
1.25M MgCl2

Solution A: 2mls Solution O
36µl ß-mercaptoethanol
10µl 100mM dATP
10µl 100mM dGTP
10µl 100mM dTTP

Solution B: Random hexamers; (Pharmacia), 90 OD Units/ml in TE

10mls 5X OLB: 2mls Solution A
5mls 2M HEPES, pH6.6
3mls Solution B.

DNA labelling;:
1) Boil DNA in 32µl (made up with water) for 5-10 minutes, then incubate the DNA at 37° for 5 minutes (this is clearly black magic, and may not be necessary).

2) Set up the reaction as follows: DNA 32µl
5X OLB 10µl
10mg/ml BSA 2µl
32P-dATP
(3000 Ci/mmol) 5µl
Klenow 1µl (2-5U)
The reaction is done at room temperature; most incorporation has ocurred by one hour, but it continues to increase till about 3-4 hours. Unlike nick translation, it does not then diminish, and if it's convenient, it's fine to leave it overnight.

3) Separate unincorporated nucleotides by your favourite method: mine is small spin columns in 0.5ml Eppendorf tubes.

4) You will find routine incorporation of between 50% and 80%, unless you used less than 20-30ng of DNA. The specific activity can reach about 3x10⁹ dpm/µg.

To label a DNA fragment in LGT agarose substitute the following for step 1 above.

1a.)Cut the band from an LGT gel and add 3mls of water for every gram of agarose (Eppendorf tubes weigh 1g). Boil for 10-15 minutes, then place at 37° for 10 minutes. Use 32µl of this mix in the normal reaction. The remaining DNA/gel mix can be frozen and reused many times (I boil it each time, but am not sure that it's necessary).

Notes:
a) The quantity of DNA: anything over about 5ng works; theoretically 20-40ng gives the hottest probe in the above reaction; over 100ng will reduce the final specific activity.
b) I have found one or two cases (out of many) where a supercoiled plasmid doesn't label well. This can be helped by a longer boiling step or, in desperation, linearising the plasmid before labelling.

Refs:
A.P. Feinberg and B. Vogelstein. Anal. Biochem. 132, 6-13 (1983)
Anal. Biochem. 137, 266-277 (1984)