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

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.