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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

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Real Time RT-PCR

Real-time reverse transcription-polymerase chain reaction, or Real-time RT-PCR, is an analytical tool used to determine the expression level of target genes. The method involves converting mRNA to complementary DNA with the help of an enzyme known as reverse transcriptase, followed by the PCR amplification of the cDNA. These two processes can be performed simultaneously in a single tube or separately as a two-step reaction.
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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

DNA polymerase preference determines PCR priming efficiency.

Wenjing Pan, Miranda Byrne-Steele, Chunlin Wang

¹HudsonAlpha Institute for Biotechnology, Huntsville, AL 35806, USA. jhan@hudsonalpha.org.

BMC Biotechnology

|February 1, 2014

Summary

This summary is machine-generated.

DNA polymerase priming bias was characterized using next-generation sequencing (NGS). This bias, influenced by primer sequence motifs, impacts amplification efficiency and guides optimal primer design for polymerase chain reaction (PCR).

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Area of Science:

Biotechnology
Molecular Biology
Genomics

Background:

Polymerase chain reaction (PCR) is a cornerstone of modern biotechnology.
Multiplex PCR reactions are known to introduce biases, with DNA polymerase implicated as a primary source.
Priming bias, specifically at the initiation of polymerization, is a key area of investigation.

Purpose of the Study:

To systematically characterize DNA polymerase priming bias.
To investigate the influence of primer sequence motifs on priming efficiencies.
To develop an improved primer-design strategy based on observed polymerase preferences.

Main Methods:

Amplification of a synthetic library with a 12-nucleotide random region using three commercial DNA polymerases.
Anchored primers with a random 3' hexamer end were employed.
Next-generation sequencing (NGS) was used to compare amplified and unamplified libraries after normalization.

Main Results:

High-throughput sequencing demonstrated significant DNA polymerase priming bias.
Specific sequence motifs at the primer's 3' end and downstream of the priming site influenced efficiency.
GC-rich motifs were preferentially selected, with variations observed between different DNA polymerase families.

Conclusions:

DNA polymerase priming bias was successfully characterized using a synthetic library and NGS.
Observed preferences were integrated into a primer-design program to optimize primer placement.
Understanding polymerase-primer interactions is crucial for minimizing amplification bias in multiplex PCR and improving primer design.