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Related Concept Videos

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...
Mismatch Repair01:36

Mismatch Repair

Overview
Improving Translational Accuracy02:07

Improving Translational Accuracy

Base complementarity between the three base pairs of mRNA codon and the tRNA anticodon is not a failsafe mechanism. Inaccuracies can range from a single mismatch to no correct base pairing at all. The free energy difference between the correct and nearly correct base pairs can be as small as 3 kcal/ mol. With complementarity being the only proofreading step, the estimated error frequency would be one wrong amino acid in every 100 amino acids incorporated. However, error frequencies observed in...
Maxam-Gilbert Sequencing01:05

Maxam-Gilbert Sequencing

In the same year as the discovery of the Sanger sequencing method, another group of scientists, Allan Maxam and Walter Gilbert, demonstrated their chemical-cleavage method for DNA sequencing. The Maxam-Gilbert method relies on using different chemicals that can cleave the DNA sequence at specific sites, the separation of resulting DNA fragments of variable size using electrophoresis, and deciphering the DNA sequence from the resulting gel bands.
Challenges of the Maxam-Gilbert Method
The...
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
Mismatch Repair01:20

Mismatch Repair

Organisms are capable of detecting and fixing nucleotide mismatches that occur during DNA replication. This sophisticated process requires identifying the new strand and replacing the erroneous bases with correct nucleotides. Mismatch repair is coordinated by many proteins in both prokaryotes and eukaryotes.
The Mutator Protein Family Plays a Key Role in DNA Mismatch Repair
The human genome has more than 3 billion base pairs of DNA per cell. Prior to cell division, that vast amount of genetic...

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Related Experiment Video

Updated: Jun 30, 2026

A Novel Saturation Mutagenesis Approach: Single Step Characterization of Regulatory Protein Binding Sites in RNA Using Phosphorothioates
11:49

A Novel Saturation Mutagenesis Approach: Single Step Characterization of Regulatory Protein Binding Sites in RNA Using Phosphorothioates

Published on: August 21, 2018

Single-base discrimination mediated by proofreading 3' phosphorothioate-modified primers.

Jia Zhang1, Kai Li

  • 1Carroll Canyon Road, Apt. 47, San Diego, CA 92126, USA.

Molecular Biotechnology
|December 12, 2003
PubMed
Summary

Proofreading DNA polymerases enhance primer extension fidelity. A new method uses these polymerases with modified primers for precise single-base discrimination in SNP assays.

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Last Updated: Jun 30, 2026

A Novel Saturation Mutagenesis Approach: Single Step Characterization of Regulatory Protein Binding Sites in RNA Using Phosphorothioates
11:49

A Novel Saturation Mutagenesis Approach: Single Step Characterization of Regulatory Protein Binding Sites in RNA Using Phosphorothioates

Published on: August 21, 2018

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Published on: June 19, 2018

DNA Sequence Recognition by DNA Primase Using High-Throughput Primase Profiling
08:04

DNA Sequence Recognition by DNA Primase Using High-Throughput Primase Profiling

Published on: October 8, 2019

Area of Science:

  • Molecular Biology
  • Genetics
  • Biochemistry

Background:

  • DNA polymerases with proofreading (3' exonuclease) activity exhibit higher fidelity in primer extension.
  • Despite their fidelity, these polymerases have not been widely applied in single nucleotide polymorphism (SNP) assays.

Purpose of the Study:

  • To develop a novel method for single-base discrimination utilizing proofreading DNA polymerases.
  • To investigate the efficacy of 3' phosphorothioate-modified primers in conjunction with proofreading polymerases for SNP detection.

Main Methods:

  • Employing a polymerase with 3' exonuclease activity.
  • Utilizing 3' phosphorothioate-modified primers for DNA synthesis.
  • Assessing primer extension efficiency across a range of annealing temperatures.

Main Results:

  • The combination of a proofreading polymerase and 3' phosphorothioate-modified primers functions as a single-base mismatch-sensitive switch.
  • DNA polymerization occurred exclusively with perfectly matched primers.
  • Mismatched primers were effectively inhibited from extension, demonstrating high specificity.

Conclusions:

  • This novel approach enables precise single-base discrimination.
  • The method shows significant potential for improving the accuracy and efficiency of SNP genotyping assays.