Ultrasensitive nucleic acid detection based on phosphorothioated hairpin-assisted isothermal amplification

Yujin Jung,Jayeon Song,Hyun Gyu Park

doi:10.1038/s41598-021-87948-8

Yujin Jung, Jayeon Song + Show 1 more

Open Access

https://doi.org/10.1038/s41598-021-87948-8

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Abstract

Herein, we describe a phosphorothioated hairpin-assisted isothermal amplification (PHAmp) method for detection of a target nucleic acid. The hairpin probe (HP) is designed to contain a 5′ phosphorothioate (PS)-modified overhang, a target recognition site, and a 3′ self-priming (SP) region. Upon binding to the target nucleic acid, the HP opens and the SP region is rearranged to serve as a primer. The subsequent process of strand displacement DNA synthesis recycles the bound target to open another HP and produces an extended HP (EP) with a PS-DNA/DNA duplex at the end, which would be readily denatured due to its reduced thermal stability. The trigger then binds to the denatured 3′ end of the EP and is extended, producing an intermediate double-stranded (ds) DNA product (IP). The trigger also binds to the denatured 3′ end of the IP, and its extension produces the final dsDNA product along with concomitant displacement and recycling of EP. By monitoring the dsDNA products, the target nucleic acid can be identified down to 0.29 fM with a wide dynamic range from 1 nM to 1 fM yielding an excellent specificity to discriminate even a single base-mismatched target. The unique design principle could provide new insights into the development of novel isothermal amplification methods for nucleic acid detection.

Highlights

We describe a phosphorothioated hairpin-assisted isothermal amplification (PHAmp) method for detection of a target nucleic acid
The following strand displacement DNA synthesis produces extended HP (EP) whose stem end is composed of a PS-DNA/DNA duplex and concomitantly recycles target nucleic acid to initiate another phase 1 reaction by binding to another free hairpin probe (HP)
The trigger binds to the denatured 3′ DNA of the IP in the same manner and is extended to produce an final dsDNA product (FP) without any PS-DNA modification, simultaneously displacing and recycling the EP to initiate another phase 2 reaction. The combination of these two reaction phases would produce a large number of EPs and FPs, which can be monitored in real-time via duplex-specific SYBR Green I staining

Summary

Introduction

We describe a phosphorothioated hairpin-assisted isothermal amplification (PHAmp) method for detection of a target nucleic acid. There is a great incentive existing to develop a more advanced isothermal method that would resolve the above-mentioned limitations while preserving the high amplification efficiency Based on this background, we describe a novel phosphorothioated hairpin-assisted isothermal amplification (PHAmp) technology capable of detecting target nucleic acids under isothermal conditions, which relies. By taking advantage of this behavior, we designed a hairpin probe (HP) that consists of a 5′ PS-DNA overhang, a target recognition site, and a 3′ self-priming (SP) region and verified that the PHAmp reaction is able to reliably identify target nucleic acid in a quite simplified manner requiring using only two simple DNA probes and a single enzyme

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