Elimination of Unspecific Amplification in the Exponential Amplification Reaction Using Polyethylene Glycol 200 (PEG 200) as a Cosolute

Abstract

Exponential amplification reaction (EXPAR) enables extremely rapid nucleic acid measurements. However, the nonspecific amplification in EXPAR limits its application, especially for low abundance target nucleic acids. Herein, polyethylene glycol 200 (PEG200), a widely available small-molecular cosolute, is described to suppress this nonspecific amplification. This cosolute generates molecular crowding and reduces the solution water activity, thereby weakening the template-template interactions and catalysis of enzymes, the major causes of nonspecific amplification. The additional of PEG200 greatly reduces the background and thereby improves the sensitivity of EXPAR by 4 orders of magnitude without extending the analysis time. Hence, the determination of target miRNAs at 1 aM was obtained within 20 min, outperforming most conventional strategies. This work provides an efficient and affordable strategy of applying water soluble cosolutes to eliminate the nonspecific amplification in EXPAR, which is expected to be widely employed to eliminate the background in nicking enzyme assisted amplification (NEAA).

Keywords:

• Cosolute
• exponential amplification reaction (EXPAR)
• polyethylene glycol 200 (PEG 200)
• unspecific amplification

Authors’ contributions

Chao Jiang, Xinguang Zhang and Yuting Shan performed the experiments; Chao Jiang and Yang Li analyzed the data; Yang Li, Cuiping Ma, and Chao Shi designed the study; Yang Li and Chao Jiang wrote the manuscript; and all authors contributed to the writing of the paper, had primary responsibility for the final content, and read and approved the final manuscript.

Disclosure statement

The authors declare that there are no competing interests associated with this manuscript.

Additional information

Funding

We highly appreciate the financial support of the Science and Technology Benefiting the People Demonstration Project of Qingdao (22-2-7-smjk-2-nsh), the Natural Science Foundation of Shandong Province (ZR2023MC064), and the Key Project of Shandong Province Natural Science Foundation (ZR2020KH030).