Tuesday, February 14, 2012

Sensitive RNA detection by combining three-way junction formation and primer generation-rolling circle amplification

A recently published report by Murakami et al. (Nucleic Acid Res. doi:10.1093/nar/gkr909) describes a novel RNA sequence-specific detection method that uses a Phi 29-based rolling-circle amplification system, with potential utility in molecular diagnostic applications. The method involves using a sequence-specific circular probe system that is synthesized using CircLigase™ ssDNA Ligase. The original method, called primer generation-rolling circle amplification (PG-RCA), was developed to detect low amounts of DNA (see Epicentre Forum 16-2, 373K PDF).

The current method combines PG-RCA with the use of three-way junction (3WJ) probes designed to hybridize to short complementary sequences on the target RNA. The probes do not interact in the absence of target RNA. Next, the addition of DNA polymerase and a "nickase" enzyme results in an isothermal cycling process to generate signal primers. These signal primers are used in the final step of PG-RCA to amplify the desired target.

The level of amplification was determined by SYBR Green dye binding. Fluorescence intensity increases as more double-stranded DNA is accumulated. A negative result (low to no fluorescence) is obtained when the circular probe is not able to hybridize to the target sequence on the RNA. The authors were able to detect 15.9 zmol (9.55 × 103 molecules) of synthetic RNA or 143 zmol (8.6 × 104 molecules) of in vitro transcribed human CD4 mRNA. The authors conclude that the 3WJ/PG-RCA technique provides an excellent means to detect very low levels of RNA expression in samples such as clinical specimens.

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