Part:BBa_K4613210

Dumbbell Template

Dumbbell Template (DT), a uniquely designed single-strand oligonucleotide, is one kind of padlock probe. Its 3'-end and phosphorylated 5'-end each contains complementary parts. After proper hybridization, the dumbbell template can be synthesized enzymatically or chemically through the intramolecular ligation of phosphate and hydroxyl end groups. Initially, the closed sealed probe maintained a stable dumbbell-shaped structure. However, when the rolling circle amplification primer bound to the toehold region, spontaneous branch migration resulted in the conversion of the dumbbell-type sealed probe into activated circular probe, thus triggering rolling circle amplification (RCA).

We used T4 DNA ligase for the reaction, next added Exo I and Exo III to degrade the excess ssDNA together with the nicked dsDNA. The agarose gel electrophoresis image of ligation product was shown as Fig. 1.

Fig. 1 (a) The principle of DT ligation; (b) Results of DT ligation.

Compared with the linear DNA padlock, the dumbbell template has the advantage of splint-free and purification-free, to reduce non-specific amplification. Owing to its flexible structure and simplicity, we selected it as our template for rolling circle amplification.

Fig.2 (a)Verification of RCA Reation using DT. M: Marker. Lane2: negative control. Lane3: 100 μM RCA primer. (b) Effect of different RCA primers' concentrations on the amount of RCA products. M: Marker. Lane2-7, different concentrations of RCA primers (0 to 100 μM).

Results of the system detected by quantitative PCR instrument were shown below.

Fig. 2 The response of the method to OTA at varied concentrations (0 pM to 20000 pM). The fluorescence values were detected by quantitative PCR instrument.

Reference

1. Liang K, Wang H, Li P, et al.Detection of microRNAs using toehold-initiated rolling circle amplification and fluorescence resonance energy transfer[J].Talanta,2020, 207: 120285.
2. Xu L, Duan J, Chen J, et al.Recent advances in rolling circle amplification-based biosensing strategies-A review[J].Anal Chim Acta,2021, 1148: 238187.
3. Zhang J, Lu Y, Gao W, et al. Structure-switching locked hairpin triggered rolling circle amplification for ochratoxin A (OTA) detection by ICP-MS[J]. Microchemical Journal, 2023, 186: 108365.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]