Rolling circle replication
The RCA mechanism is widely used in molecular biology and biomedical nanotechnology, especially in the field of biosensing (as a method of signal amplification).
These linear copies can be converted to double-stranded circular molecules through the following process: First, the initiator protein makes another nick in the DNA to terminate synthesis of the first (leading) strand.
It is believed that this replication mechanism of HPV may have physiological implications into the integration of the virus into the host chromosome and eventual progression into cervical cancer.
It is a virus that is responsible for destroying many major crops, such as cassava, cotton, legumes, maize, tomato and okra.
Rep is also strikingly similar to most other rolling replication initiator proteins of eubacteria, with the presence of motifs I, II, and III at is N terminus.
After Rep, along with other replication proteins, binds to the dsDNA it forms a stem loop where the DNA is then cleaved at the nanomer sequence causing a displacement of the strand.
[10] The derivative form of rolling circle replication has been successfully used for amplification of DNA from very small amounts of starting material.
Different from conventional DNA amplification techniques such as polymerase chain reaction (PCR), RCA is an isothermal nucleic acid amplification technique where the polymerase continuously adds single nucleotides to a primer annealed to a circular template which results in a long concatemer ssDNA that contains tens to hundreds of tandem repeats (complementary to the circular template).
One of them is the hyperbranched rolling circle amplification or HRCA, where primers that anneal to the original RCA products are added, and also extended.
[13] RCA can amplify a single molecular binding event over a thousandfold, making it particularly useful for detecting targets with ultra-low abundance.
RCA reactions can be performed in not only free solution environments, but also on a solid surface like glass, micro- or nano-bead, microwell plates, microfluidic devices or even paper strips.
In this way, RCA is becoming a highly versatile signal amplification tool with wide-ranging applications in genomics, proteomics, diagnosis and biosensing.
This technique combines two fields: RCA, which allows nucleotide amplification, and immunoassay, which uses antibodies specific to intracellular or free biomarkers.
As a result, immuno-RCA gives a specific amplified signal (high signal-to-noise ratio), making it suitable for detecting, quantifying and visualizing low abundance proteic markers in liquid-phase immunoassays[14][15][16] and immunohistochemistry.
For example, RCA has been successfully used for detecting the existence of viral and bacterial DNA from clinical samples,[20][21] which is very beneficial for rapid diagnostics of infectious diseases.
The products of RCA can also be use as templates for periodic assembly of nanospecies or proteins, synthesis of metallic nanowires[22] and formation of nano-islands.
