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Related Concept Videos

Proofreading01:31

Proofreading

Synthesis of new DNA molecules is carried out by the enzyme DNA polymerase, which adds nucleotides on the daughter strand complementary to the template DNA strand. DNA polymerase has a higher affinity to add the correct base and ensures fidelity during DNA replication. Furthermore,  it exhibits proofreading activity during replication, using an exonuclease domain that cuts off incorrect nucleotides from the nascent DNA strand.
Errors During Replication are Corrected by the DNA Polymerase Enzyme
Proofreading01:43

Proofreading

Synthesis of new DNA molecules starts when DNA polymerase links nucleotides together in a sequence that is complementary to the template DNA strand. DNA polymerase has a higher affinity for the correct base to ensure fidelity in DNA replication. The DNA polymerase furthermore proofreads during replication, using an exonuclease domain that cuts off incorrect nucleotides from the nascent DNA strand.Errors during Replication Are Corrected by the DNA Polymerase EnzymeGenomic DNA is synthesized in...
PCR - Polymerase Chain Reaction01:32

PCR - Polymerase Chain Reaction

Overview
PCR01:32

PCR

Overview
Translesion DNA Polymerases02:10

Translesion DNA Polymerases

Translesion (TLS) polymerases rescue stalled DNA polymerases at sites of damaged bases by replacing the replicative polymerase and installing a nucleotide across the damaged site. Doing so, TLS allows additional time for the cell to repair the damage before resuming regular DNA replication.
TLS polymerases are found in all three domains of life - archaea, bacteria, and eukaryotes. Of the different classes of TLS polymerases, members of the Y family are fitted with specialized structures that...
The Replisome03:01

The Replisome

DNA replication is carried out by a large complex of proteins that act in a coordinated matter to achieve high-fidelity DNA replication. Together this complex is known as the DNA replication machinery or the replisome.
The synthesis of the leading and lagging strands is a highly coordinated process. To explain this, the “Trombone model” was proposed by Bruce Alberts in 1980. The DNA loop formation starts when a primer is synthesized on the parent lagging strand. The loop grows with the...

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Related Experiment Video

Updated: Jul 2, 2026

DNA Polymerase Activity Assay Using Near-infrared Fluorescent Labeled DNA Visualized by Acrylamide Gel Electrophoresis
07:38

DNA Polymerase Activity Assay Using Near-infrared Fluorescent Labeled DNA Visualized by Acrylamide Gel Electrophoresis

Published on: October 6, 2017

DNA polymerase profiling.

Daniel Summerer1

  • 1The Scripps Research Institute, La Jolla, CA, USA.

Methods in Molecular Biology (Clifton, N.J.)
|August 13, 2008
PubMed
Summary
This summary is machine-generated.

We developed a simple, high-throughput fluorescence assay to quantify DNA polymerase activity. This method enables real-time reaction monitoring and screening of enzyme inhibitors and mutant libraries without purification.

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Last Updated: Jul 2, 2026

DNA Polymerase Activity Assay Using Near-infrared Fluorescent Labeled DNA Visualized by Acrylamide Gel Electrophoresis
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Proofreading and DNA Repair Assay Using Single Nucleotide Extension and MALDI-TOF Mass Spectrometry Analysis
11:08

Proofreading and DNA Repair Assay Using Single Nucleotide Extension and MALDI-TOF Mass Spectrometry Analysis

Published on: June 19, 2018

Area of Science:

  • Biochemistry
  • Molecular Biology
  • Assay Development

Background:

  • DNA polymerases are crucial enzymes for DNA replication and repair.
  • High-throughput screening methods are needed to study DNA polymerase function and identify inhibitors.
  • Existing assays often require multiple purification steps, limiting efficiency.

Purpose of the Study:

  • To develop a simple, homogeneous fluorescence assay for quantifying DNA polymerase activity.
  • To enable high-throughput screening of DNA polymerase function, kinetics, and inhibitors.
  • To facilitate the detection of enzymatic activity in bacterial lysates, bypassing purification.

Main Methods:

  • Utilized a molecular beacon with a template strand extension mechanism.
  • Employed Förster Resonance Energy Transfer (FRET) between two dyes for signal generation.
  • Monitored real-time fluorescence changes to quantify enzyme activity.
  • Applied the assay to two model DNA polymerases.

Main Results:

  • Successfully quantified DNA polymerase function using a homogeneous fluorescence assay.
  • Demonstrated real-time reaction profiling and kinetic characterization of DNA polymerases.
  • Showcased rapid optimization of reaction conditions and inhibitor profiling.
  • Validated detection of enzymatic activity directly in bacterial expression lysates.

Conclusions:

  • The developed fluorescence assay is a simple and effective tool for high-throughput quantification of DNA polymerase activity.
  • This assay streamlines screening processes by eliminating purification steps.
  • It facilitates kinetic studies, reaction optimization, and inhibitor screening for DNA polymerases.