- to print

DNA/RNA Modifying Enzymes

DNA Polymerase I

Product information

Features

• Incorporates modified nucleotides (e.g. biotin-, digoxigenin-, aminoallyl-, fluorescently-labeled nucleotides).
• Active in restriction enzyme, PCR and RT buffers.

Applications

• DNA labeling by nick-translation in conjunction with DNase I (1-3).
• Second-strand synthesis of cDNA in conjunction with RNase H (4).

Description

DNA Polymerase I, a template-dependent DNA polymerase, catalyzes 5'=>3' synthesis of DNA. The enzyme also exhibits 3'=>5' exonuclease (proofreading) activity, 5'=>3' exonuclease activity and ribonuclease H activity.

Source

E.coli cells with a cloned polA gene.

Molecular Weight

103 kDa monomer.

Definition of Activity Unit

One unit of the enzyme catalyzes the incorporation of 10 nmol of deoxyribonucleotides into a polynucleotide fraction (adsorbed on DE-81) in 30 min at 37°C, using poly(dA-dT)·poly(dA-dT) as a template·primer.

Enzyme activity is assayed in the following mixture: 67 mM potassium phosphate (pH 7.4), 6.7 mM MgCl₂, 1 mM 2-mercaptoethanol, 0.033 mM dATP, 0.033 mM dTTP, 0.4 MBq/ml [³H]-dTTP and 62.5 µg/ml poly(dA-dT)·poly(dA-dT).

Storage Buffer

The enzyme is supplied in:
25 mM Tris-HCl (pH 7.5), 0.1 mM EDTA, 1 mM DTT and 50% (v/v) glycerol.

10X Reaction Buffer

500 mM Tris-HCl (pH 7.5 at 25°C), 100 mM MgCl₂, 10 mM DTT.

Quality Control

The absence of endodeoxyribonucleases confirmed by appropriate quality test.

Inhibition and Inactivation

• Inhibitors: metal chelators, PP_i, P_i (at high concentrations) (5).
• Inactivated by heating at 75°C for 10 min or by addition of EDTA.

Patents, Licenses, Trademarks

Protocols & recommendations

RECOMMENDATIONS FOR USE

ADDITIONAL PROTOCOLS

II. Second Strand cDNA synthesis

Perform first strand cDNA synthesis reaction according to recommendations provided for a specific reverse transcriptase.

1. Add the following (on ice) to 20 µl of first strand cDNA synthesis reaction mixture:
   

   10X reaction buffer for DNA Polymerase I	8 µl
   RNase H	0.2 µl (1 u)
   DNA Polymerase I	3 µl (30 u)
   Water, nuclease-free	88.8 µl
   Total volume	100 µl

2. Gently vortex and briefly centrifuge.
3. Incubate at 15°C for 2 hours. Do not let the temperature rise above 15°C.
4. Add 2.5 µl (12.5 u) of T4 DNA Polymerase and incubate at 15°C for 5 min.
5. Terminate the reaction by adding 5 µl of 0.5 M EDTA, pH 8.0.

Phenol/chloroform purified blunt-end cDNA can be used for further cloning related procedures e.g. adapter ligation, phosphorylation, size fractionation, ligation and transformation.

Radioactive and Non-radioactive DNA Labeling by Nick-translation

I. Radioactive DNA labeling by nick-translation

1. Mix the following components:

   10X reaction buffer for DNA Polymerase I	2.5 µl
   Mixture of 3 dNTPs, 1 mM* (without the labeled dNTP)	1.25 µl
   [alpha-32P]-dNTP, ~110 TBq/mmol (3000 Ci/mmol)	1.85-3.7 MBq (50-100 µCi)
   DNase I, RNase-free freshly diluted to 0.002 u/µl**	1 µl
   DNA Polymerase I	0.5-1.5 µl (5-15 u)
   Template DNA	0.25 µg
   Water, nuclease-free	to 25 µl
   Total volume	25 µl

2. Immediately incubate at 15°C for 15-60 minutes.
3. Terminate the reaction by adding 1µl of 0.5 M EDTA, pH 8.0.
4. Take an aliquot (1 µl) to determin the efficiency of the label incorporation. A specific activity of DNA at least 10⁸ cpm/µg DNA is expected.
5. If needed, the labeled DNA may be separated from the unincorporated radioactive precursors on Sephadex G-50 or Bio-Gel P-60 column or using spin column (e.g. GeneJET™ PCR Purification Kit (#K0701).

Note

* To prepare a mixture of three non-labeled dNTPs (1 mM of each), mix 1 µl aliquots of stock solutions of each dNTP (100 mM, from #R0181) with 97 µl of Water, nuclease-free. These dNTP mixes can be stored at -20°C for further use.
** The DNase I, RNase-free can be diluted with the 1X reaction buffer for DNA Polymerase I.

• The reaction volumes can be scaled up or down providing that the final concentrations of the components (DNA, dNTPs, labeled dNTP) are as indicated in the protocol.
• Radioactive DNA probes with higher specific activities can be prepared using two radioactively labeled dNTPs simultaneously. In this case, the composition of the unlabeled dNTP mix should be adjusted accordingly.

II. Non-radioactive DNA labeling by nick-translation
The protocol above can be used for non-radioactive labeling by nick-translation using biotin-11-dUTP, fluorescein-12-dUTP, DIG-dUTP or aminoallyl-dUTP:

• normal dTTP is subsituted for labeled-dUTP at a molar ratio of 1:3-1:5,
• reaction time is prolonged to 1-2 hours.

References

1. Ausubel, F.M., et al., Current Protocols in Molecular Biology, vol. 1, John Wiley and Sons, Inc., Brooklyn, New York, 3.5.3-3.5.6., 1994-2005.
2. Sambrook, J., Russell, D.W., Molecular Cloning: A Laboratory Manual, Third Edition, Cold Spring Harbor laboratory, Cold Spring Harbor, N. Y., 2001.
3. Yu, H., et al., Cyanine dye dUTP analogs for enzymatic labeling of DNA probes, Nucleic Acids Res., 22, 3226-3232, 1994.
4. Gubler, U., Hoffmann, B.J., A simple and very efficient method for generating cDNA libraries, Gene, 25, 263-269, 1983.
5. Eun, H-M., Enzymology Primer for Recombinant DNA Technology, Academic Press, INC, 1996.

Related products

• AMV Reverse Transcriptase
• M-MuLV Reverse Transcriptase
• RevertAid™ Premium Reverse Transcriptase
• Maxima® Reverse Transcriptase
• RevertAid™ Reverse Transcriptase
• RevertAid™ H Minus Reverse Transcriptase
• RNase H
• DNase I, RNase-free
• Pyrophosphatase, Inorganic (from yeast)
• SensiBlot™ Plus Nylon Membrane
• dNTP Set
• dNTP Mix, 10 mM each
• Biotin-11-dUTP
• Fluorescein-12-dUTP
• Aminoallyl-dUTP
• Aminoallyl-UTP
• 0.5 M EDTA, pH 8.0
• Water, nuclease-free