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PCR

PCR

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...

Proofreading

Proofreading

Translesion DNA Polymerases

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...

DNA Isolation

DNA Isolation

DNA isolation protocols can be fast and straightforward or complex and time-consuming depending on the type and quality of DNA required for further processing. For example, plasmid DNA extraction is a bit more complicated than genomic DNA extraction because of the need for an appropriate lysis method to separate plasmid DNA from gDNA during isolation. However, for specific applications, such as long-range DNA sequencing that require a good yield of high- quality DNA samples, we need to follow...

PCR - Polymerase Chain Reaction

PCR - Polymerase Chain Reaction

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Related Experiment Video

Updated: Mar 12, 2026

Polymerase Chain Reaction: Basic Protocol Plus Troubleshooting and Optimization Strategies

Polymerase Chain Reaction: Basic Protocol Plus Troubleshooting and Optimization Strategies

Published on: May 22, 2012

Modified DNA polymerases for PCR troubleshooting.

Marta Śpibida¹, Beata Krawczyk², Marcin Olszewski¹

¹Department of Molecular Biotechnology and Microbiology, Gdańsk University of Technology, ul. G. Narutowicza 11/12, 80-233, Gdańsk, Poland.

Journal of Applied Genetics

|November 1, 2016

Summary

Polymerase chain reaction (PCR) optimization is crucial for difficult targets. Modified DNA polymerases offer solutions to common PCR challenges, enhancing experimental efficiency and reliability.

Keywords:

Chimeric DNA polymerases Fusion DNA polymerases Mutagenesis Native DNA polymerase PCR

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Area of Science:

Molecular Biology
Biochemistry

Background:

Polymerase chain reaction (PCR) is a fundamental technique in biological sciences.
Challenges in PCR include difficult DNA targets, sample inhibitors, and issues with DNA polymerase performance.
Optimization is essential for reliable and efficient PCR results.

Purpose of the Study:

To provide a guide for PCR troubleshooting based on DNA polymerase selection.
To explain the rationale behind modifying DNA polymerases.
To detail different types of DNA polymerase modifications and their impact on PCR efficiency.

Main Methods:

Review of PCR troubleshooting strategies.
Analysis of DNA polymerase properties and modifications.
Correlation of modified DNA polymerases with PCR performance.

Main Results:

Identification of common PCR inhibitors and difficult targets.
Description of various DNA polymerase modifications (e.g., engineered mutations, chemical modifications).
Demonstration of improved PCR success rates using modified polymerases for specific applications.

Conclusions:

Modified DNA polymerases are effective tools for overcoming PCR limitations.
Strategic selection and application of modified polymerases enhance PCR efficiency and broaden its applicability.
Understanding polymerase function is key to successful PCR optimization.