3 years ago

Fluorometric determination of microRNA based on strand displacement amplification and rolling circle amplification

Shiyun Ai, Huanshun Yin, Minghui Wang, Jun Wang, Yunlei Zhou, Bingchen Li

Abstract

The authors describe a fluorometric assay for microRNA. It is based on two-step amplification involving (a) strand displacement replication and (b) rolling circle amplification. The strand displacement amplification system is making use of template DNA (containing a sequence that is complementary to microRNA-21) and nicking enzyme sites. After hybridization, the microRNA strand becomes extended by DNA polymerase chain reaction and then cleaved by the nicking enzyme. The DNA thus produced acts as a primer in rolling circle amplification. Then, the DNA probe SYBR Green II is added to bind to ssDNA to generate a fluorescent signal which increases with increasing concentration of microRNA. The method has a wide detection range that covers the10 f. to 0.1 nM microRNA concentration range and has a detection limit as low as 1.0 fM. The method was successfully applied to the determination of microRNA-21 in the serum of healthy and breast cancer patients.

Graphical abstract

Schematic of a fluorometric microRNA assay based on two-step amplification involving strand displacement replication and rolling circle amplification. DNA probe SYBR Green II is then bound to ssDNA to generate a fluorescent signal which increases with increasing concentration of microRNA.

Publisher URL: https://link.springer.com/article/10.1007/s00604-017-2450-6

DOI: 10.1007/s00604-017-2450-6

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