Production of dumbbell probe through hairpin cleavage-ligation and increasing RCA sensitivity and specificity by circle to circle amplification.

Hua Wei,Suming Tang,Guojie Zhao,Tianyu Hu,Yifu Guan

doi:10.1038/srep29229

Hua Wei, Suming Tang + Show 3 more

Open Access

https://doi.org/10.1038/srep29229

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Abstract

Dumbbell probe (DP) attracts increasing interests in rolling circle amplification (RCA). A universal DP production method through cleavage-ligation of hairpin was proposed and optimized. The production is characterized by restriction endonuclease (RE)-induced cleavage ends ligation. It has the advantage of phosphorylation-free, splint-free and purification-free. To optimize designing, we found that the position of RE cleavage sequence in the stem and the primer position in the loop affected the formation and amplification of DP obviously. Both sticky and blunt ends cleaved by RE produce DP efficiently. Moreover, we introduced this DP into circle to circle (C2C) RCA based on the same cleavage-ligation principle, and acquired high sensitivity. By combining a two-ligation design and the C2C strategy, specificity for detecting let-7 family members was increased extremely. Furthermore, coreaction of different steps facilitated convenient formation and amplification process of DP.

Highlights

DNA limits its application in a mixed nucleic acids reaction system
Almost all oligonucleotides are solid-phase synthesized from 3′to 5′end[16], they are deficient in 5′phosphorate group, which is requisite for DNA ligase activity[17]
The circular template of Rolling circle amplification (RCA) is traditionally formed by the ligation of a linear template in a head-to-tail way with a requisite 5′phosphorylation

Summary

Introduction

DNA limits its application in a mixed nucleic acids reaction system. almost all oligonucleotides are solid-phase synthesized from 3′to 5′end[16], they are deficient in 5′phosphorate group, which is requisite for DNA ligase activity[17]. We presented a circular template production method based on hairpin cleavage-ligation process. We explored key factors affecting this method, including the position of RE cleavage sequence in the stem, the primer binding position in the loop, and the usage of different kinds of REs. by employing another round of cleavage-ligation, we realized circle to circle (C2C) amplification to enhance both the sensitivity and specificity of DP RCA obviously.

Results

Conclusion