Sensitive isothermal detection of nucleic-acid sequence by primer generation–rolling circle amplification

Abstract

A simple isothermal nucleic-acid amplification reaction, primer generation–rolling circle amplification (PG–RCA), was developed to detect specific nucleic-acid sequences of sample DNA. This amplification method is achievable at a constant temperature (e.g. 60°C) simply by mixing circular single-stranded DNA probe, DNA polymerase and nicking enzyme. Unlike conventional nucleic-acid amplification reactions such as polymerase chain reaction (PCR), this reaction does not require exogenous primers, which often cause primer dimerization or non-specific amplification. Instead, ‘primers’ are generated and accumulated during the reaction. The circular probe carries only two sequences: (i) a hybridization sequence to the sample DNA and (ii) a recognition sequence of the nicking enzyme. In PG–RCA, the circular probe first hybridizes with the sample DNA, and then a cascade reaction of linear rolling circle amplification and nicking reactions takes place. In contrast with conventional linear rolling circle amplification, the signal amplification is in an exponential mode since many copies of ‘primers’ are successively produced by multiple nicking reactions. Under the optimized condition, we obtained a remarkable sensitivity of 84.5 ymol (50.7 molecules) of synthetic sample DNA and 0.163 pg (∼60 molecules) of genomic DNA from Listeria monocytogenes, indicating strong applicability of PG–RCA to various molecular diagnostic assays.

Isothermal Amplification and Quantification of Nucleic Acids and its Use in Microsystems

“…Optimized RCA was able to detect 0.163 pg (~ 60 molecules) of genomic DNA from Listeria monocytogenes [149], or 143 zmol (8.6 x 10 4 molecules) of in vitro transcribed human CD4 mRNA [150].…”

Expanding Possibilities of Rolling Circle Amplification as a Biosensing Platform

“…16 This ligase catalyzes the circularization of oligonucleotide by intramolecular ligation between a phosphate at 5′-end and a hydroxyl group at the 3′-end without a guide oligonucleotide. 17 This precircularized ssDNA probe is successively treated by ssDNA-specific exonuclease, such as exonuclease I, 18,19 to degrade the linear ssDNA remaining in the ligation mixture, which causes non-specific DNA synthesis in the RCA reaction. 20 Therefore, a series of treatments including precircularization and successive degradation of the linear ssDNA probe have dramatically improved the reliability of the RCA reaction due to clear negative control.…”

“…20 Therefore, a series of treatments including precircularization and successive degradation of the linear ssDNA probe have dramatically improved the reliability of the RCA reaction due to clear negative control. [19][20][21][22][23] A lack of nonspecific amplification in test samples considerably contributes to the accuracy of RCA.…”

“…RPRCA combined with thermophilic RNase H is also achieved using thermostable Vent (exo-) DNA polymerase possessing strand displacement activity. 50 Taken together, therefore, RNase H could significantly enhance the potential towards practical uses of RPRCA in detecting RNA.…”

Simple, Colorimetric Detection of MicroRNA Based on Target Amplification and DNAzyme

“…Even though PCR (polymerase chain reaction) as a sensitive and well developed technology was commonly used for nucleic acid quantification and validation in point of care diagnosis, it faces challenges when dealing with miRNAs because of the inability of much shorter primers to efficiently bind on short miRNA templates. 19 Recently, many signal amplifying methods based on enzyme reactions (e.g., rolling circle amplification, [20][21][22][23] polymerizing/nicking amplification 24,25 ) and enzyme-free reactions 26,27 have been explored for biosensing, coupled with various nanomaterials, such as nanowire, 28 nanocluster, 29 and nanoparticles. 30 Enzyme participating methods have the advantage of high efficiency, while the enzyme-free methods outweigh their counterparts in cost.…”