Molecular Techniques and Methods

Non-Radioactive Tailing of
3'-end Oligonucleotides by Terminal Transferase

Copy Right © 2001/ Institute of Molecular Development LLC

INTRODUCTION

In this protocol, terminal transferase adds a mixture of DIG-dUTP and dATP (unlabeled) to the 3' ends of an oligonucleotide in a template-independent reaction. In the tailing reaction, the concentrations of DIG-dUTP and dATP (unlabeled) are adjusted to produce the highest hapten incorporation, optimal spacing of the hapten, and the highest sensitivity. This procedure produces a tail which ranges in length from 10-100 nucleotides (average: 50), and contains 5 DIG-dUTP molecules (on average). Alternatively, this procedure may be modified to add only 2-3 DIG-dUTP molecules, without any intervening dATP.




MATERIALS AND SOLUTIONS

5 x Reaction Buffer (1 ml)
1 M Cacodylate-sodium salt ---------------------- 160 mg
0.125 M Tris-HCl (pH 6.6) ----------------------- 125 ul of 1 M Tris-HCl
Bovine Serum Albumin ---------------------------- 1.25 mg
Add sterile H2O to make a final volume of ------- 1 ml


Purified Oligonucleotide in sterile H20

1 mM DIG-dUTP in sterile H20
(or, 1 mM Biotin-dUTP/ Fluorescein-dUTP)

10 mM dATP [in 10 mM Tris-HCl buffer (pH 7.5)]

Reaction Stop (1 ml)
0.2 M EDTA (pH 8.0) --------------------------- 400 ul of 0.5 M EDTA
Glycogen Solution (10 ug/ul) ---------------------- 10 ul
Sterile H2O -------------------------------------- 590 ul




PROCEDURES

1. Add the following components to a microcentrifuge tube on ice.

5 x Reaction Buffer --------------------------------- 4 ul
25 mM CoCl2 -------------------------------------- 4 ul
Oligonucleotide ------------------------------------- 100 pmole
1 mM DIG-dUTP ---------------------------------- 1 ul
10 mM dATP -------------------------------------- 1 ul
Terminal Transferase (50 U/ul) ---------------------- 1 ul
Add sterile H20 to make a final volume of --------- 20 ul


2. Incubate the tube at 37oC for 15 minutes.

3. Place the tube on ice.

4. Add 2 ul of Reaction Stop.

5. Add 2.5 ul 4 M LiCl and 75 ul prechilled (-20oC) 100% ethanol.

6. Let the precipitate form for at least 30 min at -70oC (or, 2 h at -20oC).

7. Centrifuge the tube (at 13,000g) for 15 min at 4oC.

8. Discard the supernatant.

9. Wash the pellet with 50 ul ice-cold 70% (v/v) ethanol.

10. Centrifuge the tube (at 13,000g) for 5 min at 4oC.

11. Discard the supernatant.

12. Dry the pellet under vacuum.

13. Dissolve the pellet in a minimal amount of sterile H20, then dilute an aliquot of the probe solution to a convenient stock concentration (e.g., 1-7 ng/ul) in the hybridization buffer to be used for the in situ experiment.

(If you are not going to use the labeled oligonucleotide probe immediately, dissolve the pellet in a minimal amount of sterile H20 and store the probe solution at -20oC.)

14. The efficiency of the tailing reaction can be checked by comparison with the direct detection.

  • The tailed oligonucleotide can be analyzed by polyacrylamide-gel electrophoresis and subsequent silver staining in comparison to the untailed oligonucleotide. DIG-tailing of oligonucleotides results in a heterogeneous shift to higher molecular weight and is detectable as a smear in polyacrylamide gels.





  • NOTES

  • HPLC- or gel-purified oligonucleotides frorn 12-100 nucleotides long can be labeled in this procedure.


  • Do not increase the concentration of oligonucleotide in this standard reaction. To make larger amounts of labeled oligonucleotide, scale up all reaction components and volumes.


  • In terminal transferase reaction, 1 mM Biotin-dUTP (or, 1 mM Fluorescein-dUTP) can be used instead of 1 mM DIG-dUTP.


  • Do not use phenol: CHCl3: IAA extraction to stop the reaction, since the labeled oligonucleotide will migrate to the organic layer during such extraction.


  • Caution: Avoid repeated freezing and thawing of the probe.



  • KIT INFORMATION




    REFERENCES

  • Schmitz, G.G., Walter, T., Seibl, R., and Kessler, C., 1991, Nonradioactive labeling of oligonucleotides in vitro with the hapten digoxigenin by tailing with terminal transferase. Anal. Biochem. 192, 222-231.


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

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