phi29 Polymerase (EN20)

phi29 Polymerase (EN20)

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phi29 Polymerase is a highly processive recombinant polymerase with exceptional strand displacement activity, therefore allows very efficient isothermal DNA amplification. The enzyme is capable of amplification of up to 70 thousands base insertions per binding event. The phi 29 polymerase possesses a 3’→5’ proofreading exonuclease activity, acting preferentially on ssDNA or RNA. Above all, extremely high yields of amplified DNA can be obtained even from minute amounts of template. Find out more about our Polymerases.
EN20-10, EN20-50

SKU: EN20 Categories: ,

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phi29 DNA Polymerase is a highly processive recombinant polymerase with exceptional strand displacement activity, which allows for highly efficient isothermal DNA amplification. The enzyme is capable of up to 70 thousands base insertions per binding event. The polymerase also possesses a 3’to 5’ proofreading exonuclease activity acting preferentially on ssDNA or RNA. Therefore 3′-modified primers are recommended.

Features

  • Recombinant polymerase derived from the Bacillus subtilis phage phi29 and over-expressed in E. coli.
  • Extremely processive polymerase, enabling amplification up to 70 kbp.
  • Extremely high yields of amplified DNA can be obtained even from minute amounts of template.
  • High-fidelity polymerase – possesses a 3’-5’ exonuclease (proofreading) activity acting preferentially on ssDNA or RNA.
  • Isothermal polymerase – no need for thermal cycling.
  • Due to heat lability of the enzyme, it is possible to use the amplification products directly in the downstream applications.

Applications

  • Rolling Circle Amplification (RCA).
  • In situ genotyping with padlock probes.
  • Amplification of DNA for SNP and STR detection.
  • Unbiased amplification of whole genome.
  • DNA template preparation for sequencing.
  • RNA-primed DNA amplification.
  • Multiple displacement amplification (MDA).
  • Cell-free amplification of DNA from single cells.
  • Amplification of DNA from environmental samples.

Unit definition

One unit of phi29 DNA Polymerase is defined as the amount of enzyme that will incorporate 0.5 pmol of dCMP into a polynucleotide fraction in 10 minutes at 30°C under standard assay conditions.

phi29 Polymerase (EN20) - Manual

Additional information

  • Enzyme Concentration: 10 U/μl
  •  Inactivated by heating at 65°C for 10 minutes.
  • Keep tubes with phi29 polymerase on ice or place in pre-chilled cooling racks while setting up the reactions.
  • The presence of active reducing reagent in the reaction buffer is critical for this enzyme. While reaction buffer supplied with the enzyme contains DTT, older buffer stocks or stocks that have been repeatedly frozen and thawed should be supplemented with 1-4 mM DTT to obtain maximal activity.

10x phi Reaction Buffer

500 mM Tris-HCl (pH 7.5 at 25°C), 100 mM (NH4)2SO4, 100 mM MgCl2, 40 mM DTT

Storage buffer

50 mM Tris-HCl (pH 7.5 at 25°C), 100 mM KCl, 1 mM DTT, 0.1 mM EDTA,
0.5% (v/v) NP-40, 0.5% (v/v) Tween 20 and 50% (v/v) glycerol.

Storage conditions

All components should be stored at -20°C in a freezer without a defrost cycle. When stored under optimum conditions, the reagents are stable until the expiry date.

Shipping conditions

Shipping on dry ice.

A: No, DNA polymerases cannot fill in the 3' overhangs.

A: No, DNA polymerases cannot remove the 5' overhangs.

A: Yes, heat at 65°C for 10 minutes.

A: Below, you can find phi29 DNA Polymerase rates at different temperatures with the use of a primed single-stranded M13 template:

(1): 2280 nt/min at 30°C; 1490 nt/min at 15°C; 290 nt/min at 4°C (1) Soengas M, Gutierrez M, Salas M. „Helix-destabilizing activity of phi 29 single-stranded DNA binding protein: effect on the elongation rate during strand displacement DNA replication”. J. Mol. Biol. 1995; 253(4): 517-529.

A: As the molecular weight of the starting material decreases, the efficiency of the amplification of MDA (multiple displacement amplification) reaction instantly diminishes, which means that the polymerase is unsuitable for amplification of low-molecular weight DNA. However, it is required for amplification processes with the use of extremely low amounts of starting DNA material, making it ideal for single cell analysis.

A: Both linear and circular DNA templates may be amplified by phi29 DNA Polymerase.

A: Yes, it is.

A: It catalyzes polymerization reaction and demonstrates three degradative activities (pyrophosphorolytic and 3`->5` DNA and 3’->5’ ssRNA exonucleolytic activities). It is important to underline that RNA hydrolysis efficiency is 10-times lower than DNA’s. The Enzyme’s exoribonuclease activity may be employed for target RNA conversion into a primer for RCA, expanding the application potential of this multifunctional enzyme and creating new opportunities for RNA detection (2). Additionally, it shows the ability to amplify RNA-containing circular substrates during RCA (reverse transcriptase activity of the phi29 DNA polymerase). Supplementing manganese ions, as a cofactor, supports replication of RNAs during RCA (3).

(2) Lagunavicius A, Kiveryte Z, Zimbaite-Ruskuliene V, Radzvilavicius T, Janulaitis A. „Duality of polynucleotide substrates for phi29 DNA polymerase: 3’-5’ RNase activity of the enzyme.” RNA 2008; (14):3 503-513. (3) Krzywkowski T, Kuhnemund M, Wu D, Nilsson M. „Limited reverse transcriptase activity of phi29 DNA polymerase.” Nucleic Acids Res. 2018; 46 (7), 3625-3632.

A: Yes. The 3′ → 5′ exonuclease activity of phi29 DNA polymerase can gradually digest the 3’-tail of target DNA even if the 3′-tail folds into an intramolecular double-stranded structure, thus it can efficiently convert different lengths of target DNA-containing fragments into RCA primers (4). In this method (exonucleolytic digestion-assisted RCA, ED-RCA) the target DNA-containing fragments triggers the formation of circular RCA template and initiates the subsequent RCA reaction.

(4) Li XY, Du YC, Zhang YP, Kong DM. „Dual functional phi29 DNA polymerase-triggered exponential rolling circle amplification for sequence-specific detection of target DNA embedded in long-stranded genomic DNA.” Sci. Rep. 2017; 7, 6263.

A: Yes, it does. However, the supplied reaction buffer that contains DTT, the reducing agent, degrades over time and should be supplemented with freshly prepared DTT for buffer stocks that are older than 4 months or those frozen and thawed more than 10 times. To replenish reducing agent add 40 μL of 1 M DTT per 1 mL of 10X reaction buffer.

A: Phi29 DNA polymerase is an enzyme commonly used in DNA amplification methods such as rolling circle amplification (RCA) and multiple strand displacement amplification (MDA), as well as in DNA sequencing methods such as single molecule real time (SMRT) sequencing.

A: If this happens, it is possible that our reaction buffer is not suitable for your application. You should test other reaction buffers.

Quality control

Polymerization activity is tested in RCA reaction with different amounts of enzyme. 3’-5’ exonuclease activity is tested by incubation of polymerase phi29 with linear oligonucleotides. DNase contamination is judged by gel electrophoresis following incubation of 1 μg of DNA with polymerase phi29 for 4 h at 37°C. DNA contamination is tested in qPCR for absence of host DNA..

phi29 Polymerase (EN20) - MSDS


phi29 Polymerase (EN20) - L. IAE805874

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phi29 Polymerase (EN20) - L. IAZ856574

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