DNA polymerases are present in all organisms and enzymes important that synthesize DNA molecules. They are used in various fields of science, especially as an important component for the in vitro synthesis of DNA, known as PCR. modern diagnostics, molecular biology and genetic engineering of DNA polymerase needs which demonstrate improved performance. This study aims to get NeqSSB-TaqS new DNA polymerase fusion of domain Stoffel Taq DNA and single-stranded DNA binding protein from Nanoarchaeum equitans as to significantly improve the properties of the DNA polymerase.
Stoffel Taq DNA coding sequence specific DNA polymerase and DNA-binding proteins Nanoarchaeum equitans (NeqSSB-like protein) fused. A new recombinant genes obtained were cloned into pET-30 Ek / LIC vector and introduced into E. coli for expression. Purified recombinant enzymes and enzymatic properties including the activity of DNA polymerase, PCR amplification level, thermostability, processivity and resistance to inhibitors, were tested. The results of the target protein around 18 mg / l after 24 hours of IPTG induction.
The specific activity of the polymerase is 2200 U / mg. Recombinant NeqSSB-TaqS exhibited a higher level extensions (1000 bp template 20 s), processivity (19 nt), thermostability (half-life 35 minutes at 95 ° C) and a higher tolerance for PCR inhibitors (0.3 to 1.25 % of whole blood, 0.84 to 13.5 ug heparin lactoferrin and 4.7 to 150 ng) of Taq DNA polymerase Stoffel. In addition, our research shows that NeqSSB-TaqS DNA polymerase has a high degree of flexibility in relation to the Mg 2+ ions (from 1 to 5 mM) and KCl or (NH4) 2SO4 salt (more than 60 mM and 40 mM, respectively ). NeqSSB-TaqS using DNA polymerase Taq DNA polymerase can not be a better choice in many applications of PCR.
Fusion of Taq DNA polymerase with single-stranded DNA binding-like protein of Nanoarchaeum equitans-Expression and characterization.
dUTPs zwitterionic conjugated with Cy3 or Cy5 analog Fluorophore effective substrate for DNA amplification and labeling by Taq polymerase.
To develop the structural modifications dNTP compatible with Taq DNA polymerase activity, we synthesized eight dUTP derivatives conjugated with Cy3 or Cy5 dyes of different analog responsible and charge distribution throughout the fluorophore. Cy3- derivatives and commercial dUTP and Cy5-dUTP studied in Taq polymerase dependent polymerase chain reaction (PCR) and the primer extension reaction using a model template that contains one, two and three adjacent nucleotides adenine.
The relative amount of DNA is amplified and kinetic parameters Km and Vmax characterize grief labeled incorporation have been estimated using fluorescence measurements and analyzed. The dUTPs label with analog zwitterionic electroneutral of Cy3 or Cy5 fluorophores Taq polymerase is used by approximately one order of magnitude more effective than dUTPs analog labeled with a negatively charged of Cy3 or Cy5. Nucleotidyl transferase activity of Taq polymerase were also observed and resulted in the addition dumps electroneutral labeled with fluorophores or positively charged to the 3 ‘end of the DNA.
Description: Intact Genomics Taq DNA Polymerase is a thermostable DNA polymerase that possesses a 5´→3´ polymerase activity (1, 2) and a 5´ flap endonuclease activity (3, 4). This product is supplied with 10x PCR reaction buffer, containing MgCl2, which produces a final Mg2+ concentration of 1.5 mM. Ideal for primary extension reaction with DNA fragments having dA overhang on 3’ ends.Product Includes:Taq DNA Polymerase10x PCR Buffer with Mg2+5x Magic Enhancer
Description: Intact Genomics Taq DNA Polymerase is a thermostable DNA polymerase that possesses a 5´→3´ polymerase activity (1, 2) and a 5´ flap endonuclease activity (3, 4). This product is supplied with 10x PCR reaction buffer, containing MgCl2, which produces a final Mg2+ concentration of 1.5 mM. Ideal for primary extension reaction with DNA fragments having dA overhang on 3’ ends.Product Includes:Taq DNA Polymerase10x PCR Buffer with Mg2+5x Magic Enhancer
Description: Intact Genomics Taq DNA Polymerase is a thermostable DNA polymerase that possesses a 5´→3´ polymerase activity (1, 2) and a 5´ flap endonuclease activity (3, 4). This product is supplied with 10x PCR reaction buffer, containing MgCl2, which produces a final Mg2+ concentration of 1.5 mM. Ideal for primary extension reaction with DNA fragments having dA overhang on 3’ ends.Product Includes:Taq DNA Polymerase10x PCR Buffer with Mg2+5x Magic Enhancer
Description: Intact Genomics Taq DNA Polymerase is a thermostable DNA polymerase that possesses a 5´→3´ polymerase activity (1, 2) and a 5´ flap endonuclease activity (3, 4). This product is supplied with 10x PCR reaction buffer, containing MgCl2, which produces a final Mg2+ concentration of 1.5 mM. Ideal for primary extension reaction with DNA fragments having dA overhang on 3’ ends.Product Includes:Taq DNA Polymerase10x PCR Buffer with Mg2+5x Magic Enhancer10mM dNTP Mix
Description: Taq DNA Polymerase is a highly thermostable DNA Polymerase that catalyzes the 5’-3’ synthesis of DNA. This polymerase has 5’-3’ exonuclease activities, lacks 3’-5’ exonuclease activity, and produces 3’-dA-tailed amplicons. PCR products made with Taq can be used with TA cloning vectors.
Description: The enzyme is supplemented with two inert gel tracking dyes (red and yellow),allowing direct loading of PCR products to agarose gels (1.0 U/μl)
Description: The enzyme is supplemented with two inert gel tracking dyes (red and yellow),allowing direct loading of PCR products to agarose gels (1.0 U/μl)
Description: The enzyme is supplemented with two inert gel tracking dyes (red and yellow),allowing direct loading of PCR products to agarose gels (1.0 U/μl)
Description: The enzyme is supplemented with two inert gel tracking dyes (red and yellow),allowing direct loading of PCR products to agarose gels (1.0 U/μl)
The introduction of mutual compensation costs to fluorophores or other functional groups dNTP conjugation can be considered as a basis for the preparation of modified nucleoside PCR-compatible triphosphates.Using Sso7d from Sulfolobus solfataricus as DNA binding protein fusion Taq DNA polymerase at the amino end, we reported hyper-expression and purification methodology novel Sso7d-Taq polymerase (S-Taq) using a two-phase system of water extraction followed by Ni-affinity chromatography.
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