Random Primer DNA labeling

DESCRIPTION

Labeling of DNA by random oligonucleotide-primed synthesis is based on the investigations of A.Feinberg and B.Vogelstein [1, 2] and is a good alternative to nick translation for producing uniformly radioactive DNA of high specific activity. The method relies on priming of the polymerase reaction on the template DNA with random hexanucleotide primers. The complementary strand is synthesized from the 3'-end of the primer with the help of DNA Polymerase I, Exonuclease Minus (Klenow Fragment, exo-) in the presence of labeled deoxyribonucleoside triphosphates. Using Klenow Fragment, exo- the reaction time can be prolonged without the fear of labeled probe degradation.

The HexaLabel™ DNA Labeling Kit offered by Fermentas is an efficient and convenient means for preparation of highly labeled DNA (>109dpm/µg) for use diverse procedures in molecular biology, such as various types of hybridization analyses [3-6]. Either dATP or dCTP could be used with the kit as a radioactive precursor.

Now, non-radioactive labeling Mix (-DIG-dUTP) is also included in the kit as an alternative labeling of DNA (DIG-dUTP is not included).

COMPONENTS OF THE KIT

1.Klenow Fragment, exo-, 5u*/µl: 15µl (30µl) of the enzyme solution in buffer containing 50% glycerol.

2.Hexanucleotide in 5X Reaction Buffer: 100µl (300µl) of 0.25M Tris-HCl buffer (pH 8.0 at 20℃) containing 25mM MgCl2, 5mM dithiotreitol and Random (hexamer) primer (7.5o.u./ml).

3.Mix A (minus dATP): 30µl (90µl) of 0.33mM dGTP, 0.33mM dTTP, 0.33mM dCTP aqueous solution.

4.Mix C (minus dCTP): 30µl (90µl) of 0.33mM dGTP, 0.33mM dATP, 0.33mM dTTP aqueous solution.

5.dNTP Mix: 40µl (120µl) of 0.25mM dGTP, 0.25mM dATP, 0.25mM dTTP, 0.25mM dCTP aqueous solution.

6.Control Template: for 2 (5) control labeling reactions, 20µl (50µl) of lambda phage DNA/HindIII fragments (10µg/ml).

7.Non-radioactive Labeling Mix (minus DIG-dUTP):50µl (150µl) of 1mM dGTP, 1mM dATP, 1mM dCTP, 0.65mM dTTP aqueous solution.

8.Deionized Water: 1.5ml of water deionized on a Milli-Q® system.

* One unit of Klenow fragment, exo- catalyzes the incorporation of 10nmoles of deoxyribonucleotides into a polynucleotide fraction (adsorbed on DE-81) in 30min at 37℃, using poly(dA-dT)•poly(dA-dT) as a template • primer.

** If you use ethanol solution of labeled [alpha-32P]-dATP or [alpha-32P]-dCTP, the needed quantity of these nucleotides dry under vacuum and redissolve in 6µl of deionized water.

EXPERIMENTAL PROTOCOL

Radioactive DNA Labeling

1.Add the following components into 1.5ml microcentrifuge tube:

Vortex the tube and spin down in a microcentrifuge for 3-5sec.

Incubate the tube in a boiling water bath for 5-10min and cool it on ice. Spin down quickly.

2.Based on your choice of labeled triphosphate (dATP or dCTP) use Mix A or Mix C, respectively.