Molecular Techniques and Methods

DNA Labeling by Random Priming

Copy Right © 2001/ Institute of Molecular Development LLC

INTRODUCTION

A mixture of random hexanucleotides is used to prime DNA synthesis in vitro from linear double-stranded template DNA. With this method, it is possible to generate probes of extremely high specific activity (routinely >109 cpm/ug), even using DNA fragments directly cut from agarose gels. Since the input DNA serves as a template and remains intact during the reaction, minimal amounts of DNA (25 ng) can be labeled to a high specific activity. Typically, greater than 70% of the labeled deoxyribonucleotide can be incorporated. The resulting probes frequently are longer than nick translated probes and can be greater than 50% of the length of the original template.




MATERIALS AND SOLUTIONS

5 x Labeling Buffer (1 ml)

250 mM Tris-HCl (pH 8.0) --------------------- 250 ul of 1 M Tris-HCl
25 mM MgCl2 ---------------------------------- 25 ul of 1 M MgCl2
10 mM DTT ------------------------------------ 10 ul of 1 M DTT
1 mM HEPES (pH 6.6) ------------------------- 10 ul of 0.1 M HEPES
Random hexadeoxyribonucleotides -------------- 26 A260 units/ml
Add distilled H2O to make a final volume of --- 1 ml
  • Store at -20oC.




    PROCEDURES

    1. Dissolve the DNA to be labeled in sterile H2O at 1-25 ng/ul.

    2. Denature the sample by heating it in a microcentrifuge tube at 100oC for 2 minutes.

    3. Rapidly chill the tube in an ice bath.

    4. Assemble the reaction in a separate microcentrifuge tube on ice by the addition of the following reagents in the order listed.

    5 x Klenow Buffer
    10 ul
    500 uM mixture of the unlabeled dNTPs without dCTP
    2 ul
    Denatured DNA template
    25 ng
    10 mg/ml acetylated (nuclease-free) BSA
    2 ul
    [a-32P]dCTP (3,000 Ci/ mmole and 10 mCi/ml)
    5 ul
    Klenow Fragment (5 U/ul)
    5 ul
    Add deionized H2O to make a final volume of
    50 ul



    3. Mix gently and incubate the reaction tube at room temperature for 1 hour.

    4. Terminate the reaction by heating at 100oC for 2 minutes and subsequently chilling in an ice bath.

    5. Add 2 ul of 0.5 M EDTA.

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

    7. Use directly in a hybridization reaction.




    NOTES

  • To achieve an optimum probe length, the amount of radiolabeled dNTP should be 10-125 pmoles. However, the highest incorporation efficiency occurs when 30 pmoles of a labeled dNTP are present. A high incorporation efficiency becomes important when the background contributed by unincorporated label is an issue.


  • The final specific activity of the DNA is influenced by two factors:
    - The specific activity of the labeled dNTP (Ci/mmole).
    - How many of the 4 dNTPs (at 10-125 pmole each) in a reaction are radiolabeled.


  • The volume of aqueous labeled dNTP should not exceed 50% of the total reaction volume. Labeled dNTPs supplied in 50% ethanol must be evaporated to dryness and redissolved in H20 before use in the reaction.



  • KIT INFORMATION




    REFERENCES

  • Feinberg, AP, Vogelstein, B (1983) Anal. Biochem., 132, 6-13.


  • Feinberg, AP, Vogelstein, B (1984) Anal. Biochem., 137, 266-267.


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

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