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Proofreading

Proofreading

Proofreading

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...

Fixing Double-strand Breaks

Fixing Double-strand Breaks

The double-stranded structure of DNA has two major advantages. First, it serves as a safe repository of genetic information where one strand serves as the back-up in case the other strand is damaged. Second, the double-helical structure can be wrapped around proteins called histones to form nucleosomes, which can then be tightly wound to form chromosomes. This way, DNA chains up to 2 inches long can be contained within microscopic structures in a cell. A double-stranded break not only damages...

Single-Strand DNA Binding Proteins

Single-Strand DNA Binding Proteins

For successful DNA replication, the unwinding of double-stranded DNA must be accompanied by stabilization and protection of the separated single strands of the DNA. This crucial task is performed by single-strand DNA-binding (SSB) proteins. They bind to the DNA in a sequence-independent manner, which means that the nitrogenous bases of the DNA need not be present in a specific order for binding of SSB proteins to it. The binding of SSB proteins straightens single-stranded DNA (ssDNA) and makes...

Mismatch Repair

Mismatch Repair

Mismatch Repair

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: Apr 8, 2026

Detection of Homologous Recombination Intermediates via Proximity Ligation and Quantitative PCR in Saccharomyces cerevisiae

Detection of Homologous Recombination Intermediates via Proximity Ligation and Quantitative PCR in Saccharomyces cerevisiae

Published on: September 11, 2022

ssDNA Pairing Accuracy Increases When Abasic Sites Divide Nucleotides into Small Groups.

Alexandra Peacock-Villada¹, Vincent Coljee¹, Claudia Danilowicz¹

¹Department of Physics, Harvard University, 17 Oxford St., Cambridge, MA 02138, United States of America.

|June 27, 2015

Summary

Introducing abasic sites into single-stranded DNA (ssDNA) enhances temperature-based discrimination between correct and mismatched base pairings. This method improves DNA hybridization accuracy for applications like gene chips and PCR.

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

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

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

Published on: June 19, 2018

Area of Science:

Molecular Biology
Biochemistry
Biophysics

Background:

Accurate sequence-dependent pairing of single-stranded DNA (ssDNA) is crucial for various molecular biology techniques, including gene chips, DNA origami, and polymerase chain reactions (PCR).
Traditional methods struggle to differentiate between perfectly matched and mismatched DNA sequences for lengths exceeding approximately 10 nucleotides, as their melting temperature differences are often less than 3°C.

Purpose of the Study:

To investigate the impact of incorporating abasic sites into ssDNA on the melting temperature differences between correct and mismatched base pairings.
To determine if abasic sites can enhance the accuracy of temperature-dependent DNA hybridization assays.

Main Methods:

Strategically grouping 35-base segments in ssDNA with the inclusion of abasic sites.
Analyzing the melting temperatures of DNA duplexes with and without abasic sites to quantify differences between matched and mismatched pairings.
Evaluating the effect of abasic sites on the cooperative melting behavior of long double-stranded DNA (dsDNA).

Main Results:

The introduction of appropriately spaced abasic sites significantly increases the melting temperature difference between correct and mismatched base pairings in ssDNA.
Mismatches in dsDNA with abasic sites destabilize annealing more effectively across the entire molecule compared to dsDNA without abasic sites.
Abasic sites promote more uniform melting of dsDNA, enhancing the overall reliability of temperature-based discrimination.

Conclusions:

The strategic placement of abasic sites in ssDNA offers a robust method to improve temperature-based discrimination between correct and incorrect base pairings.
This approach holds significant potential for advancing the accuracy and reliability of DNA-based technologies, such as diagnostic assays and synthetic biology applications.