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Author Spotlight: Development of Simplified CRISPR-Based Tests for Rapid Detection of Infectious Diseases

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An error prone PCR method for small amplicons.

Sea On Lee¹, Stephen D Fried²

¹Department of Chemistry, Johns Hopkins University, Baltimore, MD, USA.

Analytical Biochemistry

|June 3, 2021

Summary

This summary is machine-generated.

We optimized error-prone PCR (epPCR) for small DNA fragments, enhancing mutation concentration. This modified protocol is ideal for researchers needing high mutation loads in specific DNA regions.

Keywords:

Error rate Error-prone PCR Mutational frequency Mutazyme II Touchdown PCR

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Rare Event Detection Using Error-corrected DNA and RNA Sequencing

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Published on: August 3, 2018

Area of Science:

Molecular Biology
Biotechnology
Genetics

Background:

Error-prone PCR (epPCR) is vital for generating DNA mutation libraries in directed evolution.
Existing epPCR protocols are often ineffective for small DNA amplicons (<100 bp).

Purpose of the Study:

To develop a modified epPCR protocol suitable for small DNA amplicons.
To enable precise mutation concentration in targeted DNA regions.

Main Methods:

A modified error-prone PCR protocol was developed.
The protocol incorporates a Touchdown PCR approach.
Only commercially available reagents were utilized.

Main Results:

The modified epPCR protocol effectively mutagenizes small DNA amplicons (<100 bp).
The protocol allows for concentrated mutations within specific DNA regions.
High mutational loads can be achieved on standard-sized amplicons.

Conclusions:

This optimized epPCR method expands the utility of error-prone PCR for molecular biology applications.
The protocol provides a reliable tool for researchers studying gene function and protein engineering.