T4 Polynucleotide Kinase
Description:
Catalyzes the transfer and exchange of Pi from the ¦Ã position of ATP to the 5´ -hydroxyl terminus of polynucleotides (double-and single-stranded DNA and RNA) and nucleoside 3´-monophosphates. Polynucleotide Kinase also catalyzes the removal of 3´-phosphoryl groups from 3´-phosphoryl polynucleotides, deoxynucleoside 3´-monophosphates and deoxynucleoside 3´-diphosphates (1).
Source:
A E. coli strain that carries the cloned T4 Polynucleotide Kinase gene. It is purified by ion exchange chromatograph and hydrophobic chromatograph..
Applications:
End-labeling DNA or RNA for probes and DNA sequencing (2)
Addition of 5´-phosphates to oligonucleotides to allow subsequent ligation
Removal of 3´-phosphoryl groups (3)
Enzyme Properties
Heat Inactivation: 65oC for 20 minutes
Theoretical MW: 36KD
Reaction Conditions:
1X T4 PNKBuffer
Incubate at 37oC.
1X T4 PNKBuffer:
70 mM Tris-HCl
10 mM MgCl2
5 mM Dithiothreitol
pH 7.6 @ 25oC
Unit Definition:
One unit is defined as the same amount of the enzyme from a common used supplier to phosphorylate synthesized oligos to allow ligation.
Concentration:
10,000 units/ml
Storage Conditions:
10 mM Tris-HCl
50 mM KCl
0.1 uM ATP
1 mM Dithiothreitol
0.1 mM EDTA
50% Glycerol
pH 7.4 @ 25oC
Storage Temperature:
-20oC
Usage notes:
[33P] ATP may be substituted for [32P] ATP.
For radioactive labeling, use 1-50 pmol of 5´ termini in a 50 µl reaction containing 1X T4 Polynucleotide Kinase Buffer, 50 pmol of gamma-[32]P ATP and 20 units of T4 Polynucleotide Kinase.
For non-radioactive phosphorylation use up to 300 pmol of 5´ termini in a 50 µl reaction containing 1X T4 Polynucleotide Kinase Buffer, 1 mM ATP and 10 units of T4 Polynucleotide Kinase. Incubate at 37¡ãC for 30 minutes. 1X T4 DNA Ligase Buffer contains 1 mM ATP and can be substituted in non-radioactive phosphorylations (T4 Polynucleotide Kinase exhibits 100% activity in this buffer).
Fresh buffer is required for optimal activity (the reduced dithiothreitol in older buffer lowers activity).
The efficiencies of blunt and recessed 5´-end phosphorylation can be improved by heating to 70¡ãC for 5 minutes, then chilling on ice prior to kinase addition and by adding PEG-8,000 to 5% (w/v) (2).
The following levels of inhibition of T4 Polynucleotide Kinase are observed when the supplied reaction buffer is supplemented with:
50 mM NaCl - no inhibition
100 mM NaCl - 30% inhibition
150 mM NaCl - 50% inhibition
7 mM phosphate - 50% inhibition
7 mM (NH4)2SO4 - 75% inhibition
Since Polynucleotide Kinase is inhibited by ammonium ions, DNA should not be precipitated in the presence of ammonium ions prior to phosphorylation.
Dephosphorylation prior to end-labeling can be avoided by utilizing the exchange reaction, although this results in lower specific activity labelling (4). Sufficient incorporation levels can be attained using the supplied buffer supplemented with 100 uM ADP in the final reaction. However, higher levels of incorporation with the exchange reaction are achieved when using the buffer described in (2).
Gaps but not nicks can be labeled with elevated levels of ATP (1).
T4 Polynucleotide Kinase Reaction Buffer is suitable for forward reactions, but for the purpose of radiolabeling does not contain ATP. For nonradioactive phosphorylation, supplement to a final ATP concentration of 1 mM or use T4 DNA Ligase Buffer.
Endo and Exonulease Activity:
A 50 ¦Ìl reaction containing 1ug of 1kb PCR fragment and 500 units of T4 Polynucleotide Kinase incubated for 16 hours at 37oC resulted in a DNA pattern free of detectable nuclease degradation as determined by agarose gel electrophoresis.
RNase Assay:
Incubation of a 50 ul reaction containing 100 units of T4 Polynucleotide Kinase with 2 ¦Ìg of mouse total RNA for 1 hour at 37oC resulted in no detectable degradation of the RNA as determined by agarose gel electrophoresis.
References
1. Richardson, C.C. (1981) P.D. Boyer (Eds.), The Enzymes, 14, pp. 229-314. San Diego: Academic press.
2. Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, (2nd ed.), 10.59-10.67, 11.31-11.33.
3. Cameron, V. and Uhlenbeck, O.C. (1977) Biochemistry, 16, 5120-5126.
4. Berkner, K.L. and Folk, W.R. (1977) J. Biol. Chem., 252, 3176-3184
Description:
Catalyzes the transfer and exchange of Pi from the ¦Ã position of ATP to the 5´ -hydroxyl terminus of polynucleotides (double-and single-stranded DNA and RNA) and nucleoside 3´-monophosphates. Polynucleotide Kinase also catalyzes the removal of 3´-phosphoryl groups from 3´-phosphoryl polynucleotides, deoxynucleoside 3´-monophosphates and deoxynucleoside 3´-diphosphates (1).
Source:
A E. coli strain that carries the cloned T4 Polynucleotide Kinase gene. It is purified by ion exchange chromatograph and hydrophobic chromatograph..
Applications:
End-labeling DNA or RNA for probes and DNA sequencing (2)
Addition of 5´-phosphates to oligonucleotides to allow subsequent ligation
Removal of 3´-phosphoryl groups (3)
Enzyme Properties
Heat Inactivation: 65oC for 20 minutes
Theoretical MW: 36KD
Reaction Conditions:
1X T4 PNKBuffer
Incubate at 37oC.
1X T4 PNKBuffer:
70 mM Tris-HCl
10 mM MgCl2
5 mM Dithiothreitol
pH 7.6 @ 25oC
Unit Definition:
One unit is defined as the same amount of the enzyme from a common used supplier to phosphorylate synthesized oligos to allow ligation.
Concentration:
10,000 units/ml
Storage Conditions:
10 mM Tris-HCl
50 mM KCl
0.1 uM ATP
1 mM Dithiothreitol
0.1 mM EDTA
50% Glycerol
pH 7.4 @ 25oC
Storage Temperature:
-20oC
Usage notes:
[33P] ATP may be substituted for [32P] ATP.
For radioactive labeling, use 1-50 pmol of 5´ termini in a 50 µl reaction containing 1X T4 Polynucleotide Kinase Buffer, 50 pmol of gamma-[32]P ATP and 20 units of T4 Polynucleotide Kinase.
For non-radioactive phosphorylation use up to 300 pmol of 5´ termini in a 50 µl reaction containing 1X T4 Polynucleotide Kinase Buffer, 1 mM ATP and 10 units of T4 Polynucleotide Kinase. Incubate at 37¡ãC for 30 minutes. 1X T4 DNA Ligase Buffer contains 1 mM ATP and can be substituted in non-radioactive phosphorylations (T4 Polynucleotide Kinase exhibits 100% activity in this buffer).
Fresh buffer is required for optimal activity (the reduced dithiothreitol in older buffer lowers activity).
The efficiencies of blunt and recessed 5´-end phosphorylation can be improved by heating to 70¡ãC for 5 minutes, then chilling on ice prior to kinase addition and by adding PEG-8,000 to 5% (w/v) (2).
The following levels of inhibition of T4 Polynucleotide Kinase are observed when the supplied reaction buffer is supplemented with:
50 mM NaCl - no inhibition
100 mM NaCl - 30% inhibition
150 mM NaCl - 50% inhibition
7 mM phosphate - 50% inhibition
7 mM (NH4)2SO4 - 75% inhibition
Since Polynucleotide Kinase is inhibited by ammonium ions, DNA should not be precipitated in the presence of ammonium ions prior to phosphorylation.
Dephosphorylation prior to end-labeling can be avoided by utilizing the exchange reaction, although this results in lower specific activity labelling (4). Sufficient incorporation levels can be attained using the supplied buffer supplemented with 100 uM ADP in the final reaction. However, higher levels of incorporation with the exchange reaction are achieved when using the buffer described in (2).
Gaps but not nicks can be labeled with elevated levels of ATP (1).
T4 Polynucleotide Kinase Reaction Buffer is suitable for forward reactions, but for the purpose of radiolabeling does not contain ATP. For nonradioactive phosphorylation, supplement to a final ATP concentration of 1 mM or use T4 DNA Ligase Buffer.
Endo and Exonulease Activity:
A 50 ¦Ìl reaction containing 1ug of 1kb PCR fragment and 500 units of T4 Polynucleotide Kinase incubated for 16 hours at 37oC resulted in a DNA pattern free of detectable nuclease degradation as determined by agarose gel electrophoresis.
RNase Assay:
Incubation of a 50 ul reaction containing 100 units of T4 Polynucleotide Kinase with 2 ¦Ìg of mouse total RNA for 1 hour at 37oC resulted in no detectable degradation of the RNA as determined by agarose gel electrophoresis.
References
1. Richardson, C.C. (1981) P.D. Boyer (Eds.), The Enzymes, 14, pp. 229-314. San Diego: Academic press.
2. Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, (2nd ed.), 10.59-10.67, 11.31-11.33.
3. Cameron, V. and Uhlenbeck, O.C. (1977) Biochemistry, 16, 5120-5126.
4. Berkner, K.L. and Folk, W.R. (1977) J. Biol. Chem., 252, 3176-3184
