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Thermophilic DNA Polymerases

Bsm DNA Polymerase, Large Fragment

Product information

Features

• Thermophilic DNA polymerase with strong strand displacement activity.

Applications

• Isothermal DNA amplification by the method of:
  – loop-mediated isothermal amplification (LAMP) (1, 2),
  – whole genome amplification (WGA) (3),
  – ramification amplification (RAM) (4).
• Random-primed DNA labeling.
• Labeling by fill-in 5’-overhangs of dsDNA.

Description

Bsm DNA Polymerase, Large Fragment, is a portion of DNA polymerase of Bacillus smithii, which catalyzes 5'=>3' synthesis of DNA and lacks 5'=>3' and 3'=>5' exonuclease activities. Bsm DNA Polymerase, Large Fragment, has a strong strand displacement activity and is active in a wide range of temperatures from 30°C to 63°C, with an optimum of activity at 60°C. Bsm DNA Polymerase, Large Fragment, is an enzyme with high functional similarity to Bst DNA Polymerase, Large Fragment, and can replace it in most applications.

Not suitable for use in PCR.

Source

E.coli cells with a cloned polA gene from Bacillus smithii.

Molecular Weight

67 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 60°C.

Enzyme activity is assayed in the following mixture: 10 mM Tris-HCl (pH 7.5), 10 mM MgCl₂, 100 µg/ml BSA, 0.75 mM activated salmon milt DNA, 0.2 mM of each dNTP, 0.4 MBq/ml [H³]-dTTP.

Storage Buffer

The enzyme is supplied in:
10 mM Tris-HCl (pH 7.5), 50 mM KCl, 1 mM DTT, 0.1 mM EDTA, 0.15% (v/v) Triton X-100, 50% (v/v) glycerol.

10X Bsm Buffer

200 mM Tris-HCl (pH 8.8 at 25°C), 100 mM KCl, 100 mM (NH₄)₂SO₄, 20 mM MgSO₄, 1% (v/v) Tween 20.

Quality Control

The absence of endodeoxyribonucleases, exodeoxyribonucleases, ribonucleases and E.coli genomic DNA is confirmed by appropriate quality test.

Inhibition and Inactivation

Inactivated by heating at 80°C for 10 min.

Notice

Use of this enzyme in certain applications may be covered by patents and may require a license.

Patents, Licenses, Trademarks

Protocols & recommendations

RECOMMENDATIONS FOR USE

ADDITIONAL PROTOCOLS

DNA 3'-end Labeling by Fill-in of 5'-overhangs with Klenow Fragment* or Bsm DNA Polymerase, Large Fragment

1. Prepare the following reaction mixture:

   Linear DNA (aqueous solution)	0.1-4 µg
   10X reaction buffer for Klenow Fragment or 10X Bsm buffer	2 µl
   [alpha-32P]-dNTP, ~15-30 TBq/mmol (400-800 Ci/mmol) or [alpha-32P]-dNTP, ~110 TBq/mmol (3000 Ci/mmol)	0.74 MBq (20 µCi) 2.96 MBq (80 µCi)
   3 dNTP Mix, 2 mM each (without a labeled dNTP)	2.5 µl (0.25 mM final concentration)
   Klenow Fragment or Klenow Fragment, exo- or Bsm DNA Polymerase, Large Fragment	0.1 µl (1 u) 0.2 µl (1 u) 0.125 µl (1 u)
   Water, nuclease-free	to 20 µl
   Total volume	20 µl

2. Incubate at 37°C for 15 min.
3. Stop the reaction by heating at 75°C for 10 min.

Note

* This protocol is suitable labeling of the following Fermentas DNA markers, composed of DNA fragments with 5'-overhangs:
Lambda DNA EcoRI Marker, #SM028
Lambda DNA HindIII Marker, #SM0101
Lambda DNA EcoRI/HindIII Marker, #SM0191
Lambda DNA Eco91I Marker, #SM0111
phiX174 DNA HinfI Marker, #SM0261

• The modified version of this protocol can be used for non-radioactive labeling of DNA markers. Substitute a part of dTTP nucleotide with a modified nucleotide (e.g. Biotin-11-dUTP or Fluorescein-12-dUTP) at a molar ratio of 1:2.
• For estimation of pmol of DNA ends, see REviewer™.

Radioactive Random-primed DNA Labeling with Klenow Fragment, exo- or Bsm DNA Polymerase, Large Fragment

1. Prepare the following reaction mixture:

   DNA (aqueous solution)	10 µl (100 ng)
   10X reaction buffer for Klenow Fragment, exo- or 10X Bsm buffer	5 µl
   6.0 A260units/ml (100 µM) Random Hexamer Primer	12.5 µl
   Water, nuclease-free	to 40 µl
   Total volume	40 µl

2. Incubate the mixture in a boiling water bath for 5-10 minutes and then chill on ice.
3. Add:

   3 dNTP Mix, 0.33 mM each (without a labeled dNTP)	3 µl (0.02 mM final concentration)
   [alpha-32P]-dNTP, ~110 TBq/mmol (3000 Ci/mmol)	1.85 MBq (50 µCi)
   Klenow Fragment, exo- or Bsm DNA Polymerase, Large Fragment	1 µl (5 u) 1 µl (8 u)
   Water, nuclease-free	to 50 µl
   Total volume	50 µl

4. Incubate the reaction mixture for 10 minutes at 37°C.
5. Add 4 µl 0.25 mM dNTP mix and incubate at 37°C for 5 minutes.
6. Add 1 µl 0.5 M EDTA, pH 8.0 to stop the reaction.
7. Remove 1 µl of the reaction mixture and determine the percentage of label incorporated.
8. Purify by using Sephadex G-50 or Bio-Gel P-60.

References

1. Tsugunori Notomi, et al., Loop-mediated isothermal amplification of DNA, Nucleic Acids Res., v. 28, No. 12, e63, 2000.
2. Masaki Imai, et al., Rapid diagnosis of H5N1 avian influenza virus infection by newly developed influenza H5 hemaglutinin gene-specific loop-mediated isothermal amplification method, Journal of Virological Methods, 141, 173-180, 2007.
3. Dean, F.B., et al., Comprehensive human genome amplification using multiple displacement amplification, Proc. NatI. Acad. Sci. USA, 99, 5261-5266, 2002.
4. Jizu Yi, et al., Molecular Zipper: a fluorescent probe for real-time isothermal DNA amplification, Nucleic Acids Res., v.34, No.11, e81, 2006.

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