Technical aspects of nicking enzyme assisted amplification

The Analyst
Liu WangJian Wu

Abstract

Nicking enzyme assisted amplification (NEAA) is an extremely rapid method for molecular diagnosis. However, this technology is not widely applied for real sample analysis because the overproduced non-specific products limit its sensitivity and raise the threshold of detection methods. Here, we have found that the non-specific amplification is mainly caused by the coexistence of Bst polymerase, nicking primers and dNTP. The highly active nicking enzyme directs and accelerates the non-specific amplification in a way which favors nicking. To suppress the non-specific amplification, the nicking enzyme concentration, reaction temperature, and magnesium ion concentration are optimized. The compatibility of Bst polymerase with the concentration of the monovalent cation is also crucial. Besides, the sensitivity could be enhanced by shortening the target sequences and priming the 3' end of the target.

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Citations

Jul 2, 2020·Critical Reviews in Clinical Laboratory Sciences·Vinoth Kumar RajendranAnwar Sunna
May 9, 2021·Clinical Microbiology and Infection : the Official Publication of the European Society of Clinical Microbiology and Infectious Diseases·Constance DuboisJérémie F Cohen
Sep 3, 2021·Analytical Methods : Advancing Methods and Applications·Harindi JayakodyHannah S Leese

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Methods Mentioned

BETA
transgenic
PCR
electrophoresis

Software Mentioned

EXPAR
Primer Premier
NEAR
ETSeq
NUPACK

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