Molecular Techniques and Methods

DNA Labeling by Nick Translation

Copy Right © 2001/ Institute of Molecular Development LLC

INTRODUCTION

Free 3'-hydroxyl ends (nicks) are created within the unlabeled duplex DNA by DNase I. DNA polymerase I then catalyzes the addition of a nucleotide residue to the 3'-hydroxyl terminus of the nick. At the same time, the 5' to 3' exonuclease activity of this enzyme removes the nucleotide from the 5'-phosphoryl terminus of the nick. A new nucleotide with a 3'-OH group is incorporated at the position where the original nucleotide was excised, and the nick is thus shifted along by one nucleotide in a 3' direction. This 3' shift of the nick results in the sequential addition of radioactively labeled nucleotides to the DNA while the pre-existing nucleotides are removed. DNA probes prepared by nick translation can be used for a wide variety of hybridization techniques such as gel blots and colony plaque lifts. Using the protocol provided, typically greater than 65% of the labeled deoxyribonucleotide is incorporated, generating probes of high specific activity (routinely 108cpm/ug).




MATERIALS AND SOLUTIONS

10 x Nick Translation Buffer (1 ml)
500 mM Tris-HCl (pH7.2) ------------------------- 500 ul of 1 M Tris-HCl
100 mM MgSO4 ---------------------------------- 100 ul of 1 M MgSO4
1 mM DTT ---------------------------------------- 1 ul of 1 M DTT
Deionized H2O ------------------------------------ 399 ul


Stop Solution (1 ml)
0.2 M EDTA (pH8.0) ------------------------------ 400 ul of 0.5 M EDTA
Deionized H2O ------------------------------------- 600 ul




PROCEDURES

1. Mix the following components in a microfuge tube.

500 uM Nucleotide mix without dCTP
10 ul
10 x Nick Translation Buffer
5 ul
Sample DNA in H2O
1 ug
[a-32P]dCTP (70 uCi at 400 Ci/ mmole and 10 mCi/ ml)
7 ul
DNase I (0.1 U/ul)
0.5-1 ul
DNA polymerase I (10 u/ul)
2 ul
Add deionized H2O to make a final volume of
50 ul


2. Incubate at 16oC for 1 hour.

3. Add 5 ul of the Stop Solution.

4. Remove unincorporated [a-32P]dCTP.




NOTES

  • To produce probes with higher specific activities, a second labeled nucleotide (i.e., [a-32P]dATP) can be used in place of one of the unlabeled nucleotides. The final concentration of any labeled nucleotide should not fall below 3 uM.


  • It is important to maintain the reaction temperature at 16oC. Higher temperatures can produce "snapback" DNA structures which lower hybridization efficiency.


  • In general, longer incubation times result in greater incorporation of label but tend to slightly reduce the overall length of the labeled fragment.



  • KIT INFORMATION



    REFERENCES



  • Please send your comment on this protocol to "editor@MolecularInfo.com".

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