Phosphorylation of 5' prominence DNA molecules by exchange reaction

Summary

The T4 phage polynucleotide kinase-catalyzed exchange reaction (Van de Sande et al. 1973; Berkner and Folk 1977) is a rapid method for labeling 5' end DNA. Unlike the forward reaction, it does not require DNA dephosphorylation. The forward reaction is performed in a slightly acidic (pH 6.4) imidazole buffer to stimulate enzymatic dephosphorylation (Berkner and Folk 1977). The optimal Km values for the exchange reaction (300 μmol/L for ADP and 10 μmol/L for ATP) were all higher than those readily achieved in the test tubes, and thus labeling efficiencies rarely exceeded 30%. However, the exchange reaction efficiency was greatly improved by the addition of spermidine and polyethylene glycol to the reaction mixture (Lillehauget al. 1976; Harrison and Zimmol/Lmerman 1986a). This experiment is based on the "Guide to Molecular Cloning Experiments, Third Edition", translated by Huang Peitang et al.

Operation method

Phosphorylation of DNA molecules at the 5' protruding end by exchange reaction

Principle

The T4 phage polynucleotide kinase-catalyzed exchange reaction (Van de Sande et al. 1973; Berkner and Folk 1977) is a rapid method for labeling 5' end DNA. Unlike the forward reaction, it does not require DNA dephosphorylation. The forward reaction is performed in a slightly acidic (pH 6.4) imidazole buffer to stimulate enzymatic dephosphorylation (Berkner and Folk 1977). The optimal Km values for the exchange reaction (300 μmol/L for ADP and 10 μmol/L for ATP) were all higher than those readily achieved in the test tubes, and thus labeling efficiencies rarely exceeded 30%. However, the efficiency of the exchange reaction was greatly improved by the addition of spermidine and polyethylene glycol to the reaction mixture (Lillehauget al. 1976; Harrison and Zimmol/Lmerman 1986a).

Materials and Instruments

T4 Phage polynucleotide kinase DNA
ADP Ammonium acetate ATP EDTA Ethanol Imidazole buffer Polyethylene glycol
Liquid Scintillation Counters Sephadex G-50 Centrifuge Columns Sephadex G-50 Columns

Move

I. Materials

1. Buffers and solutions

ADP (1 mmol/L)

Ammonium acetate (10 mol/L)

ATP (10 mmol/L)

EDTA ( 0.5 mol/L, pH 8.0)

Ethanol

10X imidazole buffer (500 mmol/L imidazole hydrochloride (pH 6.4), 180 mmol/L MgCl2, 50 mmol/L DTT, 1 mmol/L spermidine hydrochloride, 1 mmol/L EDTA (pH 8.0))

Polyethylene glycol (24% m/V PEG 8000, formulated in water)

2. enzymes and buffers

T4 phage polynucleotide kinase

3. Nucleic acids and oligonucleotides

DNA (10 to 50 pmol m 5'-phosphorylated ends)

4. Radioactive compounds

[ ^γ-32P ] ATP ( 10 mCi/ml, specific activity 3000~7000 Ci/mmol)

5. Specialized equipment

Liquid scintillation counter, able to quantify ^32P by Cherenkov radiation

Sephadex G-50 column, equilibrated with TE (pH 7.6)

or

Sephadex G-50 column (1 ml) equilibrated with TE (pH 7.6)

ii. Methods

1. Mix in a microcentrifuge tube in the following order:

DNA containing 5' terminal phosphate 10~50 pmol

10X Imidazole Buffer 5 μl

1 mmol/L ADP 5 μl

50 nmol/L ATP 1 μl

10 mCi/ml [ ^γ-32P ] ATP

(Specific activity 3000~7000 Ci/mmol) 20~100 pmol

Water Add to 40 μl

24% (m/V) PEG 10 μl

T4 Phage polynucleotide kinase (20 units) 1 μl

Flick the outside of the tube to mix the components and incubate the reaction at 37°C for 30 min.

2. Add 2 μl of 0.5 mol/L EDTA (pH 8.0) to terminate the reaction. Detect the total activity in the reaction mixture by Cherenkov counting with a liquid scintillation counter.

3. Separate the radiolabeled probe from the undoped dNTP by any of the following methods:

Chromatography on a Sephadex G-50 centrifugal column.

or

conventional size exclusion chromatography on a 1 ml Sephadex G-50 column (equilibrated with TE)

or

Two rounds of selective precipitation of radiolabeled DNA with ammonium acetate and ethanol

4. Determine the amount of radioactivity in the probe product by Cherenkov counting and calculate the efficiency of the transfer of the radiolabel to the 5' end by dividing the amount of radioactivity in the probe by the total amount of radioactivity in the reaction mixture.

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