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A dumbbell probe-mediated rolling circle amplification strategy for highly sensitive microRNA detection.

Zhou Y, Huang Q, Gao J, Lu J, Shen X, Fan C - Nucleic Acids Res. (2010)

Bottom Line: The femtomolar sensitivity of D-RCA compares favorably with other existing technologies.More significantly, the dynamic range of D-RCA is extremely large, covering eight orders of magnitude.We also demonstrate miRNA quantification with this highly sensitive and inexpensive D-RCA strategy in clinical samples.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Physical Biology, Shanghai Institute of Applied Physics, Chinese Academy of Sciences, Shanghai, China.

ABSTRACT
We herein report the design of a dumbbell-shaped DNA probe that integrates target-binding, amplification and signaling within one multifunctional design. The dumbbell probe can initiate rolling circle amplification (D-RCA) in the presence of specific microRNA (miRNA) targets. This D-RCA-based miRNA strategy allows quantification of miRNA with very low quantity of RNA samples. The femtomolar sensitivity of D-RCA compares favorably with other existing technologies. More significantly, the dynamic range of D-RCA is extremely large, covering eight orders of magnitude. We also demonstrate miRNA quantification with this highly sensitive and inexpensive D-RCA strategy in clinical samples.

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The D-RCA strategy for miRNA detection. A dumbbell probe contains three domains, an MBD, an SGBD and a loop domain. The binding of miRNA to MBD initiates RCA in the presence of T4 DNA ligase and phi29 polymerase, which generates a long DNA sequence that contains many SGBD for an amplified fluorescent readout.
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Figure 1: The D-RCA strategy for miRNA detection. A dumbbell probe contains three domains, an MBD, an SGBD and a loop domain. The binding of miRNA to MBD initiates RCA in the presence of T4 DNA ligase and phi29 polymerase, which generates a long DNA sequence that contains many SGBD for an amplified fluorescent readout.

Mentions: Several novel methods for miRNA detection by using padlock probe-based RCA have been reported (29,30). Jonstrup et al. (29) first reported an RCA-based protocol for miRNA detection, which nevertheless relied on time-consuming northern blotting. Cheng et al. (30) improved this method by introducing a second primer, and then RCA products were analyzed via fluorescence from an intercalative dye, SYBR Green I (SG). In addition to the sensitivity, selectivity is the other important factor in miRNA detection. Several previous reports have employed a stem–loop primer (31), LNA modification (24) and high-stringency enzymes (30) to improve the specificity of miRNA detection. In this work, we propose a dumbbell probe (32)-mediated RCA (D-RCA) strategy for high sensitivity and selective miRNA detection (Figure 1). We aim to improve the sensitivity of miRNA detection via RCA, and the selectivity by using the dumbbell probe to reduce non-specific amplification.Figure 1.


A dumbbell probe-mediated rolling circle amplification strategy for highly sensitive microRNA detection.

Zhou Y, Huang Q, Gao J, Lu J, Shen X, Fan C - Nucleic Acids Res. (2010)

The D-RCA strategy for miRNA detection. A dumbbell probe contains three domains, an MBD, an SGBD and a loop domain. The binding of miRNA to MBD initiates RCA in the presence of T4 DNA ligase and phi29 polymerase, which generates a long DNA sequence that contains many SGBD for an amplified fluorescent readout.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2926626&req=5

Figure 1: The D-RCA strategy for miRNA detection. A dumbbell probe contains three domains, an MBD, an SGBD and a loop domain. The binding of miRNA to MBD initiates RCA in the presence of T4 DNA ligase and phi29 polymerase, which generates a long DNA sequence that contains many SGBD for an amplified fluorescent readout.
Mentions: Several novel methods for miRNA detection by using padlock probe-based RCA have been reported (29,30). Jonstrup et al. (29) first reported an RCA-based protocol for miRNA detection, which nevertheless relied on time-consuming northern blotting. Cheng et al. (30) improved this method by introducing a second primer, and then RCA products were analyzed via fluorescence from an intercalative dye, SYBR Green I (SG). In addition to the sensitivity, selectivity is the other important factor in miRNA detection. Several previous reports have employed a stem–loop primer (31), LNA modification (24) and high-stringency enzymes (30) to improve the specificity of miRNA detection. In this work, we propose a dumbbell probe (32)-mediated RCA (D-RCA) strategy for high sensitivity and selective miRNA detection (Figure 1). We aim to improve the sensitivity of miRNA detection via RCA, and the selectivity by using the dumbbell probe to reduce non-specific amplification.Figure 1.

Bottom Line: The femtomolar sensitivity of D-RCA compares favorably with other existing technologies.More significantly, the dynamic range of D-RCA is extremely large, covering eight orders of magnitude.We also demonstrate miRNA quantification with this highly sensitive and inexpensive D-RCA strategy in clinical samples.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Physical Biology, Shanghai Institute of Applied Physics, Chinese Academy of Sciences, Shanghai, China.

ABSTRACT
We herein report the design of a dumbbell-shaped DNA probe that integrates target-binding, amplification and signaling within one multifunctional design. The dumbbell probe can initiate rolling circle amplification (D-RCA) in the presence of specific microRNA (miRNA) targets. This D-RCA-based miRNA strategy allows quantification of miRNA with very low quantity of RNA samples. The femtomolar sensitivity of D-RCA compares favorably with other existing technologies. More significantly, the dynamic range of D-RCA is extremely large, covering eight orders of magnitude. We also demonstrate miRNA quantification with this highly sensitive and inexpensive D-RCA strategy in clinical samples.

Show MeSH