Isothermal BRCA Assay

 

Branched Rolling Circle Amplification Assay Detection Limit (LLOQ)

The rolling circle amplification assay is a non-exponential, isothermal enrichment of a target sequence by a phi29 DNA polymerase. This kind of a liquid phase ligand binding assay implies a circular DNA padlock probe, which contains the target sequence to be detected (e.g. a microRNA). If the T4 RNA Ligase 2 is applied to generate the DNA padlock probe (1), both the 5´- and the 3´-end can anneal to a target sequence (e.g. a microRNA), and the circle is afterwards closed upon ligation. Another DNA primer is then annealed to the circular DNA padlock template to initiate DNA polymerization. As the template is circular, the amplification (theoretically) proceeds until it is stopped (e.g. by heat inactivation). The target sequence (in this case, of the microRNA) is then detected by SYBR Green fluorescence.

For this kind of liquid phase microRNA binding assay Cheng et al (1) reported an lower limit of quantification (LLOQ) of approx. 0.18 pg/mL (with let-7a microRNA possessing a molecular mass of 7100 Da).

Just to compare with another microRNA liquid phase detection assay: the microRNA let-7b mimic is currently being developed by Mirna/Asuragen for lung cancer treatment (2). The complete microRNA therapeutics pipeline can be viewed here. The Mirna scientists applied the sensitive Real-Time RT-qPCR assay to the quantification of let-7b microRNA mimics in biological samples (2), which seems to be incredibly more sensitive than the rolling circle assay (assuming only 7 molecules can be detected; I saved myself the exact calculation here …). Certainly the let-7 microRNA family Real-Time RT-qPCR assay can be sequence specific, even at the single nucleotide level (3).

A very recent paper improved the rolling circle amplification assay detection limit by measuring the pyrophosphate (PPi) released by the DNA polymerase rather than the polymerization product (4), but they never reached the excellent detection limit of the Real-Time RT-qPCR assay.

 

References:
(1) Cheng Y et al (2009), Angew Chem Int Ed 48: 3268-72.
(2) Trang P et al (2011), Mol Ther, doi:10.1038/mt.2011.48.
(3) Chen C et al (2005), Nucleic Acids Res 33(29): e179.
(4) Mashimo Y et al (2911), Anal Bioanal Chem, doi 10.1007/s00216-011-5083-3.

 

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