Rolling circle replication

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Rolling circle replication describes an unidireksial nucleic acid replication process that can quickly synthesize multiple copies of DNA molecules or circular RNAs, such as plasmids, bacteriophage genes, and circular RNA genomes from viroids. Some eukaryotic viruses also replicate their DNA or RNA through a revolving circle mechanism.

As a simplified version of natural rolling circle replication, isothermal DNA amplification techniques, rolling milling circle (RCA) developed. RCA mechanisms are widely used in molecular biology & amp; biomedical nanotechnology, particularly in the field of biosensing (as a signal strengthening method).

Video Rolling circle replication

Circular DNA Replication

The replication of DNA replication is initiated by an initiator protein encoded by plasmid DNA or bacteriophage, which carries one strand of double-stranded circular DNA molecule at a site called a double strand origin, or DSO. The initiator protein remains attached to the tip of the phosphate 5 'of the entangled strand, and the free hydroxyl 3' tip is released to serve as a primer for DNA synthesis by DNA polymerase III. Using unnecessary strands as templates, replication takes place around circular DNA molecules, replacing the strand that is infected as single-stranded DNA. The stranded strand displacement is performed by a hosted encoded helicase called PcrA (abbreviated abbreviation for a reduced plasmid copy) in the presence of a plasmid replication initiation protein.

Advanced DNA synthesis can produce many single linear copies of the original DNA in a sustainable head-to-tail series called a concatemer. This linear copy can be converted into double-stranded circular molecules through the following process:

First, the initiator protein makes another nick in DNA to terminate the synthesis of the first strand (leading). RNA polymerase and DNA polymerase III then replicate the DNA of a single strand origin (SSO) to make another double circle loop. DNA polymerase I removes the primer, replaces it with DNA, and the DNA ligase joins with the edges to make another molecule of double-stranded circular DNA.

As a summary, replicating a typical loop of DNA has five steps:

1. The dsDNA circle will be "clipped".
2. End 3 'extends using "unknown" DNA "as the template strand; Edge 5 'moved.
3. The missing DNA is a thread that is left behind and made double-stranded through a series of Okazaki fragments.
4. Replication of "ssDNA" not needed "and refugees".
5. Displaced circulating DNA.

Maps Rolling circle replication

Virology

Virus DNA replication

Some DNA viruses replicate their genomic information in host cells through replicated circular replication. For example, human-6 herpes virus (HHV-6) (hibv) expresses a set of "early genes" that are believed to be involved in this process. The length of the concatemers whose results are then cleaved between pac-1 and pac-2 regions of the HHV-6 genome by ribozymes when packed into individual virions.

Human Papillomavirus-16 (HPV-16) is another virus that uses revolving replication to produce progeny at high levels. HPV-16 infects human epithelial cells and has a double-stranded circular genome. During replication, at the origin, E1 hexamer wraps a single strand of DNA and moves in the 3 'to 5' direction. In a normal two-way replication, two replication proteins will separate at the time of the collision, but on HPV-16 it is believed that E1 hexamer does not separate, causing replication to be continuous. It is believed that this mechanism of HPV replication may have physiological implications into the integration of the virus into the host chromosome and its development eventually becomes cervical cancer.

In addition, geminivirus also utilizes replicated circular replication as its replication mechanism. It is a virus that is responsible for destroying many major crops, such as cassava, cotton, beans, corn, tomatoes and okra. This virus has a circular, single-stranded DNA, which replicates in host cell cells. The entire process is initiated by a geminiviral replication initiator protein, Rep., Which is also responsible for altering the host environment to act as part of the replicating machine. The rep is also very similar to most protein initiators of rolling replication of eubacteria, with the presence of motifs I, II, and III on the N terminus. During replication of rolling circles, geminivirus ssDNA is converted to dsDNA and Rep is then attached to dsDNA in the order of origin of TAATATTAC. After Rep, along with other replication proteins, binding to dsDNA forms a loop of stem in which the DNA is subsequently cleaved in the nanomer sequence that causes strand displacement. This displacement allows fork replication to advance in the 3 'to 5' direction which eventually results in a new ssDNA strand and a concatameric strand of DNA.

Bacteriophage T4 Intermediation DNA replication includes circular and circular circular concatemeric structures. These structures tend to reflect the mechanism of the circle of replicating scrolling.

Viral RNA Replication

Some RNA and viroid viruses also replicate their genome through replicating circular RNA replication. For viroids, there are two alternative RNA replication pathways, each followed by members of the Pospivirodae family (asymmetric replication) and Avsunviroidae (symmetric replication).

In the Pospiviroidae family (PSTVd-like), circular plus RNA strands are transcribed by the host RNA polymerase into a strand of minus oligomers and then oligomers plus strands. This plus oligomeric strand is cleaved by the RNase host and ligated by the host's RNA ligand to reform the monomeric circular strands plus the strand. This is called an asymmetrical path of circular rolling replication. Viroids in the Avsunviroidae family (ASBVd-like) replicate their genome via a symmetrical path of revolving circular circulation. In this symmetrical path, the oligomeric minus strand is first split and ligated to form a monomeric minus strand, and then transcribed into the oligomeric plus strand. This plus oligomeric strand is then cleaved and ligated to reform the plus monomeric strands. The symmetric replication path is named because the plus and minus strands are produced in the same way.

The cleavage of the plus and minus oligomeric fractions is mediated by the split hammerhead ribozyme structure present in Avsunviroidae, but the structure is absent in Pospiviroidae.

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Scrolling circle amplification

The derivation of revolving circular replication has been successfully used for DNA amplification of a very small amount of starting material. This amplification technique is named as Rolling circle amplification (RCA). Unlike conventional DNA amplification techniques such as polymerase chain reaction (PCR), RCA is an isothermal nucleic acid amplification technique where polymerase continuously adds single nucleotides to primers which are annealed onto a circular template that produces long ssDNA concatemer containing tens to hundreds of tandem repeats circular templates).

There are five important components needed to perform an RCA reaction:

1. DNA polymerase
2. An appropriate buffer compatible with polymerase.
3. A short primary DNA or RNA
4. Circular DNA template
5. Deoxynucleotide triphosphates (dNTPs)

The polymerases used in RCA are Phi29, Bst, and Vent exo-DNA polymerase for DNA amplification, and T7 RNA polymerase for RNA amplification. Since Phi29 DNA polymerase has the best strand removal and transferability among all the previously mentioned polymers, it has been most commonly used in RCA reactions. Different from polymerase chain reaction (PCR), RCA can be performed at constant temperature (room temperature up to 37C) in both free solution and above immobilized target (solid phase amplification).

There are usually three steps involved in the RCA DNA reaction:

1. Circular template ligation, which can be done through template-mediated enzymatic ligation (eg, T4 DNA ligase) or template free ligation using special DNA ligases (ie, CircLigase).
2. Single stranded DNA retrieval induced by Primary. Some primers can be used for hybridization with the same circle. As a result, several amplification events can begin, resulting in some RCA products ("Multiprimed RCA"). Linear RCA products can be converted into multiple circles using restriction enzyme digestion followed by template-mediated enzymatic ligation.
3. Detection and visualization of amplification products, most commonly done through fluorescent detection, with dNTP-conjugated fluorophore, fluorophore-tethered auxiliary or fluorescently-labeled beacon molecules. In addition to fluorescent approaches, gel electrophoresis is also widely used to detect RCA products.

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RCA Application

RCA can strengthen a single molecule binding event more than a thousandfold, making it very useful for detecting targets with ultra-low abundance. RCA reactions can be performed not only in a free solution environment, but also on solid surfaces such as glass, micro or nano-manic, microwell plates, microfluidic devices or even pieces of paper. This feature makes it a very powerful tool for amplifying signals in solid phase immunoassays (eg, ELISA). In this way, RCA becomes a highly versatile signal amplification tool with extensive applications in genomics, proteomics, diagnosis and biosensing.

Immuno-RCA

Immuno-RCA is an isothermal signal amplification method for high specificity & amp; detection and quantification of proteins with high sensitivity. The technique combines two fields: RCA, which allows nucleotide amplification, and immunoassay, which use special antibodies for intracellular or free biomarkers. As a result, immuno-RCA provides a special amplified signal (high signal-to-noise ratio), making it suitable for detecting, measuring and visualizing low abundance protein markers in liquid phase immunoassays and immunohistochemicals.

Immuno-RCA follows a typical immune reacting reaction in ELISA or immunohistochemical tissue staining. The detection antibody used in the immuno-RCA reaction was modified by attaching the ssDNA oligonucleotide at the end of the heavy chain. Thus the Fab (Fragment, antigen binding) section of the detecting antibody can still bind to specific antigens and the oligonucleotides may serve as primers of the RCA reaction.

The typical immuno-RCA-mediated antibody procedures are as follows:

1. Detection antibodies recognize specific target proteins. These antibodies are also attached to the oligonucleotide primer.

2. When the circular DNA is present, it is annealed, and the primer matches with the complementary sequence of circular DNA.

3. The complementary sequence of circular DNA templates is copied hundreds of times and remains attached to antibodies.

4. RCA output (extended ssDNA) is detected by fluorescent probe using fluorescent microscope or plate reader.

Aptamer based on immuno-RCA

In addition to mediated-RCA antibodies, RCA ssDNA primers can be conjugated to the 3 'end of an aptamer DNA as well. The primary tail can be amplified through the amplification of a rolling circle. Products can be visualized through labeling of fluorescent journalists. The process is illustrated in the picture on the right.

Other RCA applications

Various RCA derivatives are widely used in the field of biosensing. For example, RCA has been successfully used to detect the presence of viral and bacterial DNA from clinical samples, which is very useful for rapid diagnosis of infectious diseases. It has also been used as an on-chip signal amplification method for microarray (for DNA and RNA) microarray testing.

In addition to the amplification functions in biosensing applications, RCA techniques can be applied to the construction of DNA nanostructures and DNA hydrogels as well. RCA products can also be used as templates for the assembly of nanospecies or proteins on a regular basis, the synthesis of metallic nanowires and the formation of nano-islands.

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References

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External links

• the DNA replication system is used with small circular DNA molecules Genome 2 , T. Brown et al., at NCBI Books
• MicrobiologyBytes: Viroid and Virusoids
• http://mcmanuslab.ucsf.edu/node/246

Source of the article : Wikipedia