Polymerase Chain Reaction-Dynamic Light Scattering Sensor for DNA and Protein by Using Both Replication and Cleavage Properties of Taq Polymerase.
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Polymerase Chain Reaction-Dynamic Light Scattering Sensor for DNA and Protein by Using Both Replication and Cleavage Properties of Taq Polymerase.
Merging AuNPs to polymerase chain reaction (PCR) has become a promising strategy to develop a sensitive sensing platform, since the desired optical properties of AuNPs and the power of exponential PCR amplification. However, the combination of AuNPs for PCR typically fail to achieve the expected sensitivity along with additional measures. Here we report a single step and universal PCR-based biosensor for detection depending on the size of nucleic acids and proteins by using the technique of dynamic light scattering (DLS).
The sensing system employs a modified DNA AuNPs (AuNPs-DNA) to serve as a TaqMan probe as the probe signal, in which the DNA on the surface of AuNPs can be parsed by Taq polymerase during PCR extension, causing aggregation of AuNPs to be detected by DLS. This strategy allows the recognition of the target-specific sequences of double-stranded DNA (dsDNA), cope with changes in background signal that was triggered by the increase of PCR cycles. Further demonstrating its application in the detection of foodborne pathogens Listeria monocytogenes with a detection limit of 1.2 fg uL-1, which is more sensitive than colorimetric methods.
In addition, by utilizing the proximity test strategy, this biosensor demonstrated the ability to detect homogeneous proteins, which indicates the detection limits for the model thrombin 13:00. Together, the work shows a reliable strategy to combine AuNPs in the PCR amplification process as the probe signal, rather than a process of post-PCR.
Polymerase Chain Reaction-Dynamic Light Scattering Sensor for DNA and Protein by Using Both Replication and Cleavage Properties of Taq Polymerase.
recombinant novel properties Sso7d-Taq DNA polymerase purified using a two-phase extraction of water: The utility of the enzyme in the diagnosis of the virus.
Using Sso7d from Sulfolobus solfataricus as DNA binding protein fusion Taq DNA polymerase at the amino end, we reported hyper-expression and purification of novel methodologies Sso7d-Taq polymerase (S-Taq) using a two-phase system of water extraction followed by Ni-affinity chromatography.
The fusion enzyme usefulness in carrying out human gene PCR directly from whole blood and in the detection of hepatitis B virus from clinical samples is shown in this article. We present data on improved thermo-stability of the S-Taq DNA polymerase over Taq DNA polymerase and also provide evidence of high stability with detergent in comparison with Taq polymerase. S-protein purified Taq shows the acceptable limit of the level of the host genomic DNA without the use of DNA accelerate DNases and other agents and show promising potential for use in diagnostic PCR-based, in-situ PCR and forensic science.
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.
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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.
