Rolling circle replication

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Rolling circle replication produces multiple copies of a single circular template. Rolling circle.svg
Rolling circle replication produces multiple copies of a single circular template.

Rolling circle replication (RCR) is a process of unidirectional nucleic acid replication that can rapidly synthesize multiple copies of circular molecules of DNA or RNA, such as plasmids, the genomes of bacteriophages, and the circular RNA genome of viroids. Some eukaryotic viruses also replicate their DNA or RNA via the rolling circle mechanism.

Contents

As a simplified version of natural rolling circle replication, an isothermal DNA amplification technique, rolling circle amplification was developed. The RCA mechanism is widely used in molecular biology and biomedical nanotechnology, especially in the field of biosensing (as a method of signal amplification). [1]

Circular DNA replication

Illustration of rolling circle replication. Rolling-circle replication.png
Illustration of rolling circle replication.

Rolling circle DNA replication is initiated by an initiator protein encoded by the plasmid or bacteriophage DNA, which nicks one strand of the double-stranded, circular DNA molecule at a site called the double-strand origin, or DSO. The initiator protein remains bound to the 5' phosphate end of the nicked strand, and the free 3' hydroxyl end is released to serve as a primer for DNA synthesis by DNA polymerase III. Using the unnicked strand as a template, replication proceeds around the circular DNA molecule, displacing the nicked strand as single-stranded DNA. Displacement of the nicked strand is carried out by a host-encoded helicase called PcrA (the abbreviation standing for plasmid copy reduced) in the presence of the plasmid replication initiation protein.

Continued DNA synthesis can produce multiple single-stranded linear copies of the original DNA in a continuous head-to-tail series called a concatemer. 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. RNA polymerase and DNA polymerase III then replicate the single-stranded origin (SSO) DNA to make another double-stranded circle. DNA polymerase I removes the primer, replacing it with DNA, and DNA ligase joins the ends to make another molecule of double-stranded circular DNA.

As a summary, a typical DNA rolling circle replication has five steps: [2]

  1. Circular dsDNA will be "nicked".
  2. The 3' end is elongated using "unnicked" DNA as leading strand (template); 5' end is displaced.
  3. Displaced DNA is a lagging strand and is made double stranded via a series of Okazaki fragments.
  4. Replication of both "unnicked" and displaced ssDNA.
  5. Displaced DNA circularizes.

Virology

Replication of viral DNA

Some DNA viruses replicate their genomic information in host cells via rolling circle replication. For instance, human herpesvirus-6 (HHV-6)(hibv) expresses a set of "early genes" that are believed to be involved in this process. [3] The long concatemers that result are subsequently cleaved between the pac-1 and pac-2 regions of HHV-6's genome by ribozymes when it is packaged into individual virions. [4]

A model for HPV16 rolling circle replication. Rolling Circle Replication Virology.jpg
A model for HPV16 rolling circle replication.

Human Papillomavirus-16 (HPV-16) is another virus that employs rolling replication to produce progeny at a high rate. HPV-16 infects human epithelial cells and has a double stranded circular genome. During replication, at the origin, the E1 hexamer wraps around the single strand DNA and moves in the 3' to 5' direction. In normal bidirectional replication, the two replication proteins will disassociate at time of collision, but in HPV-16 it is believed that the E1 hexamer does not disassociate, hence leading to a continuous rolling replication. 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. [5]

In addition, geminivirus also utilizes rolling circle replication as its replication mechanism. It is a virus that is responsible for destroying many major crops, such as cassava, cotton, legumes, maize, tomato and okra. The virus has a circular, single stranded, DNA that replicates in host plant cells. The entire process is initiated by the geminiviral replication initiator protein, Rep, which is also responsible for altering the host environment to act as part of the replication machinery. 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. During the rolling circle replication, the ssDNA of geminivirus is converted to dsDNA and Rep is then attached to the dsDNA at the origin sequence TAATATTAC. 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. This displacement allows the replication fork to progress in the 3’ to 5’ direction which ultimately yields a new ssDNA strand and a concatameric DNA strand. [6]

Bacteriophage T4 DNA replication intermediates include circular and branched circular concatemeric structures. [7] These structures likely reflect a rolling circle mechanism of replication.

Replication of viral RNA

Some RNA viruses and viroids also replicate their genome through rolling circle RNA replication. For viroids, there are two alternative RNA replication pathways that respectively followed by members of the family Pospivirodae (asymmetric replication) and Avsunviroidae (symmetric replication).

Rolling circle replication of viral RNA Rolling circle replication of RNA viroids.jpg
Rolling circle replication of viral RNA

In the family Pospiviroidae (PSTVd-like), the circular plus strand RNA is transcribed by a host RNA polymerase into oligomeric minus strands and then oligomeric plus strands. [8] These oligomeric plus strands are cleaved by a host RNase and ligated by a host RNA ligase to reform the monomeric plus strand circular RNA. This is called the asymmetric pathway of rolling circle replication. The viroids in the family Avsunviroidae (ASBVd-like) replicate their genome through the symmetric pathway of rolling circle replication. [9] In this symmetric pathway, oligomeric minus strands are first cleaved and ligated to form monomeric minus strands, and then are transcribed into oligomeric plus strands. These oligomeric plus strands are then cleaved and ligated to reform the monomeric plus strand. The symmetric replication pathway was named because both plus and minus strands are produced the same way.

Cleavage of the oligomeric plus and minus strands is mediated by the self-cleaving hammerhead ribozyme structure present in the Avsunviroidae, but such structure is absent in the Pospiviroidae. [10]

Rolling circle amplification

The molecular mechanism of Rolling Circle Amplification (RCA) Rolling circle amplification mechanism.jpg
The molecular mechanism of Rolling Circle Amplification (RCA)

The derivative form of rolling circle replication has been successfully used for amplification of DNA from very small amounts of starting material. [1] This amplification technique is named as Rolling circle amplification (RCA). 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). [11]

There are five important components required for performing a RCA reaction:

  1. A DNA polymerase
  2. A suitable buffer that is compatible with the polymerase.
  3. A short DNA or RNA primer
  4. A circular DNA template
  5. Deoxynucleotide triphosphates (dNTPs)
The detection methods of RCA product Rolling circle amplification detection.jpg
The detection methods of RCA product

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 processivity and strand displacement ability among all aforementioned polymerases, it has been most frequently used in RCA reactions. Different from polymerase chain reaction (PCR), RCA can be conducted at a constant temperature (room temperature to 65C) in both free solution and on top of immobilized targets (solid phase amplification).

There are typically three steps involved in a DNA RCA reaction:

  1. Circular template ligation, which can be conducted via template mediated enzymatic ligation (e.g., T4 DNA ligase) or template-free ligation using special DNA ligases (i.e., CircLigase).
  2. Primer-induced single-strand DNA elongation. Multiple primers can be employed to hybridize with the same circle. As a result, multiple amplification events can be initiated, producing multiple RCA products ("Multiprimed RCA").
  3. Amplification product detection and visualization, which is most commonly conducted through fluorescent detection, with fluorophore-conjugated dNTP, fluorophore-tethered complementary or fluorescently-labeled molecular beacons. In addition to the fluorescent approaches, gel electrophoresis is also widely used for the detection of RCA product.

RCA produces a linear amplification of DNA, as each circular template grows at a given speed for a certain amount of time. To increase yield and achieve exponential amplification as PCR does, several approaches have been investigated. 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. [12] In this way the original RCA creates more template that can be amplified. Another is circle to circle amplification or C2CA, where the RCA products are digested with a restriction enzyme and ligated into new circular templates using a restriction oligo, followed by a new round of RCA with a larger amount of circular templates for amplification. [13]

Applications of RCA

illustration of immuno-RCA Wiki fig2.png
illustration of immuno-RCA

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. This feature makes it a very powerful tool for amplifying signals in solid-phase immunoassays (e.g., ELISA). In this way, RCA is becoming a highly versatile signal amplification tool with wide-ranging applications in genomics, proteomics, diagnosis and biosensing.

Immuno-RCA

Immuno-RCA is an isothermal signal amplification method for high-specificity & high-sensitivity protein detection and quantification. 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.

Immuno-RCA follows a typical immuno-adsorbent reaction in ELISA or immunohistochemistry tissue staining. [17] The detection antibodies used in immuno-RCA reaction are modified by attaching a ssDNA oligonucleotide on the end of the heavy chains. So the Fab (Fragment, antigen binding) section on the detection antibody can still bind to specific antigens and the oligonucleotide can serve as a primer of the RCA reaction.

The typical antibody mediated immuno-RCA procedure is as follows:

Illustration of aptamer based immuno-rca Illustration of aptamer based immuno-rca.png
Illustration of aptamer based immuno-rca

1. A detection antibody recognizes a specific proteic target. This antibody is also attached to an oligonucleotide primer.

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

3. The complementary sequence of the circular DNA template is copied hundreds of times and remains attached to the antibody.

4. RCA output (elongated ssDNA) is detected with fluorescent probes using a fluorescent microscope or a microplate reader.

Aptamer based immuno-RCA [18]

In addition to antibody mediated immuno-RCA, the ssDNA RCA primer can be conjugated to the 3' end of a DNA aptamer as well. The primer tail can be amplified through rolling circle amplification. The product can be visualized through the labeling of fluorescent reporter. [19] The process is illustrated in the figure on the right.

Other applications of RCA

Various derivatives of RCA were widely used in the field of biosensing. 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. It has also been used as an on-chip signal amplification method for nucleic acid (for both DNA and RNA) microarray assay. [1]

In addition to the amplification function in biosensing applications, RCA technique can be applied to the construction of DNA nanostructures and DNA hydrogels as well. 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. [1]

See also

Related Research Articles

DNA virus Virus that has DNA as its genetic material

A DNA virus is a virus that has a genome made of deoxyribonucleic acid (DNA) that is replicated by a DNA polymerase. They can be divided between those that have two strands of DNA in their genome, called double-stranded DNA (dsDNA) viruses, and those that have one strand of DNA in their genome, called single-stranded DNA (ssDNA) viruses. dsDNA viruses primarily belong to two realms: Duplodnaviria and Varidnaviria, and ssDNA viruses are almost exclusively assigned to the realm Monodnaviria, which also includes dsDNA viruses. Additionally, many DNA viruses are unassigned to higher taxa. Viruses that have a DNA genome that is replicated through an RNA intermediate by a reverse transcriptase are separately considered reverse transcribing viruses and are assigned to the kingdom Pararnavirae in the realm Riboviria.

DNA replication Biological process

In molecular biology, DNA replication is the biological process of producing two identical replicas of DNA from one original DNA molecule. DNA replication occurs in all living organisms acting as the most essential part for biological inheritance. This is essential for cell division during growth and repair of damaged tissues, while it also ensures that each of the new cells receives its own copy of the DNA. The cell possesses the distinctive property of division, which makes replication of DNA essential.

Polymerase chain reaction Laboratory technique to multiply a DNA sample for study

Polymerase chain reaction (PCR) is a method widely used to rapidly make millions to billions of copies of a specific DNA sample, allowing scientists to take a very small sample of DNA and amplify it to a large enough amount to study in detail. PCR was invented in 1983 by the American biochemist Kary Mullis at Cetus Corporation. It is fundamental to many of the procedures used in genetic testing and research, including analysis of ancient samples of DNA and identification of infectious agents. Using PCR, copies of very small amounts of DNA sequences are exponentially amplified in a series of cycles of temperature changes. PCR is now a common and often indispensable technique used in medical laboratory research for a broad variety of applications including biomedical research and criminal forensics.

Primer (molecular biology)

A primer is a short single-stranded nucleic acid used by all living organisms in the initiation of DNA synthesis. DNA polymerase enzymes are only capable of adding nucleotides to the 3’-end of an existing nucleic acid, requiring a primer be bound to the template before DNA polymerase can begin a complementary strand. Living organisms use solely RNA primers, while laboratory techniques in biochemistry and molecular biology that require in vitro DNA synthesis usually use DNA primers, since they are more temperature stable.

Reverse transcriptase An enzyme which generates DNA

A reverse transcriptase (RT) is an enzyme used to generate complementary DNA (cDNA) from an RNA template, a process termed reverse transcription. Reverse transcriptases are used by viruses such as HIV and hepatitis B to replicate their genomes, by retrotransposon mobile genetic elements to proliferate within the host genome, and by eukaryotic cells to extend the telomeres at the ends of their linear chromosomes. Contrary to a widely held belief, the process does not violate the flows of genetic information as described by the classical central dogma, as transfers of information from RNA to DNA are explicitly held possible.

Viroids are small infectious pathogens. They are composed solely of a short strand of circular, single-stranded RNA. Unlike viruses, they have no protein coating. All known viroids are inhabitants of angiosperms, and most cause diseases, whose respective economic importance to humans varies widely.

Virusoids are circular single-stranded RNA(s) dependent on viruses for replication and encapsidation. The genome of virusoids consist of several hundred (200–400) nucleotides and does not code for any proteins.

DNA polymerase I

DNA polymerase I is an enzyme that participates in the process of prokaryotic DNA replication. Discovered by Arthur Kornberg in 1956, it was the first known DNA polymerase. It was initially characterized in E. coli and is ubiquitous in prokaryotes. In E. coli and many other bacteria, the gene that encodes Pol I is known as polA. The E. coli Pol I enzyme is composed of 928 amino acids, and is an example of a processive enzyme — it can sequentially catalyze multiple polymerisation steps without releasing the single-stranded template. The physiological function of Pol I is mainly to support repair of damaged DNA, but it also contributes to connecting Okazaki fragments by deleting RNA primers and replacing the ribonucleotides with DNA.

Helicase-dependent amplification (HDA) is a method for in vitro DNA amplification that takes place at a constant temperature.

<i>Geminiviridae</i> Family of viruses

Geminiviridae is a family of plant viruses that encode their genetic information on a circular genome of single-stranded (ss) DNA. There are 520 species in this family, assigned to 14 genera. Diseases associated with this family include: bright yellow mosaic, yellow mosaic, yellow mottle, leaf curling, stunting, streaks, reduced yields. They have single-stranded circular DNA genomes encoding genes that diverge in both directions from a virion strand origin of replication. According to the Baltimore classification they are considered class II viruses. It is the largest known family of single stranded DNA viruses.

Baltimore classification Virus classification system made by David Baltimore

Baltimore classification is a system used to classify viruses based on their manner of messenger RNA (mRNA) synthesis. By organizing viruses based on their manner of mRNA production, it is possible to study viruses that behave similarly as a distinct group. Seven Baltimore groups are described that take into consideration whether the viral genome is made of deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), whether the genome is single- or double-stranded, and whether the sense of a single-stranded RNA genome is positive or negative.

Non-cellular life Life that has no cellular structure

Non-cellular life, or acellular life is life that exists without a cellular structure for at least part of its life cycle. Historically, most (descriptive) definitions of life postulated that a living organism must be composed of one or more cells, but this is no longer considered necessary, and modern criteria allow for forms of life based on other structural arrangements.

RNA-dependent RNA polymerase

RNA-dependent RNA polymerase (RdRp) or RNA replicase is an enzyme that catalyzes the replication of RNA from an RNA template. Specifically, it catalyzes synthesis of the RNA strand complementary to a given RNA template. This is in contrast to typical DNA-dependent RNA polymerases, which all organisms use to catalyze the transcription of RNA from a DNA template.

Nucleic acid sequence-based amplification, commonly referred to as NASBA, is a method in molecular biology which is used to produce multiple copies of single stranded RNA. NASBA is a two-step process that takes RNA and anneals specially designed primers, then utilizes an enzyme cocktail to amplify it.

The Avsunviroidae are a family of viroids. There are four species in three genera. They consist of RNA genomes between 246–375 nucleotides in length. They are single-stranded covalent circles and have intramolecular base pairing. All members lack a central conserved region.

Hepatitis B virus DNA polymerase

Hepatitis B virus DNA polymerase is a hepatitis B viral protein. It is a DNA polymerase that can use either DNA or RNA templates and a ribonuclease H that cuts RNA in the duplex. Both functions are supplied by the reverse transcriptase (RT) domain.

Φ29 DNA polymerase is an enzyme from the bacteriophage Φ29. It is being increasingly used in molecular biology for multiple displacement DNA amplification procedures, and has a number of features that make it particularly suitable for this application. It was discovered and characterised by Spanish scientist Margarita Salas.

Reverse Transcription Loop-mediated Isothermal Amplification

Reverse transcription loop-mediated isothermal amplification (RT-LAMP) is a one step nucleic acid amplification method to multiply specific sequences of RNA. It is used to diagnose infectious disease caused by RNA viruses.

<i>Ground squirrel hepatitis virus</i> Species of virus

Ground squirrel hepatitis virus, abbreviated GSHV, is a partially double-stranded DNA virus that is closely related to human Hepatitis B virus (HBV) and Woodchuck hepatitis virus (WHV). It is a member of the family of viruses Hepadnaviridae and the genus Orthohepadnavirus. Like the other members of its family, GSHV has high degree of species and tissue specificity. It was discovered in Beechey ground squirrels, Spermophilus beecheyi, but also infects Arctic ground squirrels, Spermophilus parryi. Commonalities between GSHV and HBV include morphology, DNA polymerase activity in genome repair, cross-reacting viral antigens, and the resulting persistent infection with viral antigen in the blood (antigenemia). As a result, GSHV is used as an experimental model for HBV.

<i>Monodnaviria</i> Realm of viruses

Monodnaviria is a realm of viruses that includes all single-stranded DNA viruses that encode an endonuclease of the HUH superfamily that initiates rolling circle replication of the circular viral genome. Viruses descended from such viruses are also included in the realm, including certain linear single-stranded DNA (ssDNA) viruses and circular double-stranded DNA (dsDNA) viruses. These atypical members typically replicate through means other than rolling circle replication.

References

  1. 1 2 3 4 Ali, M. Monsur; Li, Feng; Zhang, Zhiqing; Zhang, Kaixiang; Kang, Dong-Ku; Ankrum, James A.; Le, X. Chris; Zhao, Weian (2014). "Rolling circle amplification: a versatile tool for chemical biology, materials science and medicine". Chemical Society Reviews. 43 (10): 3324–41. doi:10.1039/C3CS60439J. PMID   24643375.
  2. ↑ Demidov, Vadim V, ed. (2016). Rolling Circle Amplification (RCA) - Toward New Clinical | Vadim V. Demidov | Springer. Springer. doi:10.1007/978-3-319-42226-8. ISBN   9783319422244. S2CID   30024718.
  3. ↑ Arbuckle, Jesse (2011). "The molecular biology of human herpesvirus-6 latency and telomere integration". Microbes and Infection. 13 (8–9): 731–741. doi:10.1016/j.micinf.2011.03.006. PMC   3130849 . PMID   21458587.
  4. ↑ Borenstein, Ronen; Frenkel, Niza (2009). "Cloning human herpes virus 6A genome into bacterial artificial chromosomes and study of DNA replication intermediates". Proceedings of the National Academy of Sciences. 106 (45): 19138–19143. Bibcode:2009PNAS..10619138B. doi: 10.1073/pnas.0908504106 . PMC   2767366 . PMID   19858479.
  5. ↑ Kusumoto-Matsuo, Rika; Kanda, Tadahito; Kukimoto, Iwao (2011-01-01). "Rolling circle replication of human papillomavirus type 16 DNA in epithelial cell extracts". Genes to Cells. 16 (1): 23–33. doi:10.1111/j.1365-2443.2010.01458.x. ISSN   1365-2443. PMID   21059156. S2CID   30493728.
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  7. ↑ Bernstein H, Bernstein C (July 1973). "Circular and branched circular concatenates as possible intermediates in bacteriophage T4 DNA replication". J. Mol. Biol. 77 (3): 355–61. doi:10.1016/0022-2836(73)90443-9. PMID   4580243.
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  11. ↑ Ali, M. Monsur; Li, Feng; Zhang, Zhiqing; Zhang, Kaixiang; Kang, Dong-Ku; Ankrum, James A.; Le, X. Chris; Zhao, Weian (2014-05-21). "Rolling circle amplification: a versatile tool for chemical biology, materials science and medicine". Chemical Society Reviews. 43 (10): 3324–3341. doi:10.1039/c3cs60439j. ISSN   1460-4744. PMID   24643375.
  12. ↑ Lizardi, Paul M.; Huang, Xiaohua; Zhu, Zhengrong; Bray-Ward, Patricia; Thomas, David C.; Ward, David C. (July 1998). "Mutation detection and single-molecule counting using isothermal rolling-circle amplification". Nature Genetics. 19 (3): 225–232. doi:10.1038/898. ISSN   1546-1718. PMID   9662393. S2CID   21007563.
  13. ↑ Dahl, Fredrik; Banér, Johan; Gullberg, Mats; Mendel-Hartvig, Maritha; Landegren, Ulf; Nilsson, Mats (2004-03-30). "Circle-to-circle amplification for precise and sensitive DNA analysis". Proceedings of the National Academy of Sciences. 101 (13): 4548–4553. doi: 10.1073/pnas.0400834101 . ISSN   0027-8424. PMC   384784 . PMID   15070755.
  14. ↑ Schweitzer, Barry; Roberts, Scott; Grimwade, Brian; Shao, Weiping; Wang, Minjuan; Fu, Qin; Shu, Quiping; Laroche, Isabelle; Zhou, Zhimin (April 2002). "Multiplexed protein profiling on microarrays by rolling-circle amplification". Nature Biotechnology. 20 (4): 359–365. doi:10.1038/nbt0402-359. ISSN   1087-0156. PMC   2858761 . PMID   11923841.
  15. ↑ Zhou, Long; Ou, Li-Juan; Chu, Xia; Shen, Guo-Li; Yu, Ru-Qin (2007). "Aptamer-Based Rolling Circle Amplification: A Platform for Electrochemical Detection of Protein". Analytical Chemistry. 79 (19): 7492–7500. doi:10.1021/ac071059s. PMID   17722881.
  16. ↑ Björkesten, Johan; Patil, Sourabh; Fredolini, Claudia; Lönn, Peter; Landegren, Ulf (2020-05-29). "A multiplex platform for digital measurement of circular DNA reaction products". Nucleic Acids Research. 48 (13): gkaa419. doi:10.1093/nar/gkaa419. ISSN   0305-1048. PMC   7367203 . PMID   32469060.
  17. ↑ Gusev, Y.; Sparkowski, J.; Raghunathan, A.; Ferguson, H.; Montano, J.; Bogdan, N.; Schweitzer, B.; Wiltshire, S.; Kingsmore, S. F. (July 2001). "Rolling circle amplification: a new approach to increase sensitivity for immunohistochemistry and flow cytometry". The American Journal of Pathology. 159 (1): 63–69. doi:10.1016/S0002-9440(10)61674-4. ISSN   0002-9440. PMC   1850404 . PMID   11438455.
  18. ↑ Zhao, Weian; Ali, M. Monsur; Brook, Michael A.; Li, Yingfu (2008-08-11). "Rolling Circle Amplification: Applications in Nanotechnology and Biodetection with Functional Nucleic Acids". Angewandte Chemie International Edition. 47 (34): 6330–6337. doi:10.1002/anie.200705982. ISSN   1521-3773. PMID   18680110.
  19. ↑ Zhou, Long; Ou, Li-Juan; Chu, Xia; Shen, Guo-Li; Yu, Ru-Qin (2007-10-01). "Aptamer-Based Rolling Circle Amplification: A Platform for Electrochemical Detection of Protein". Analytical Chemistry. 79 (19): 7492–7500. doi:10.1021/ac071059s. ISSN   0003-2700. PMID   17722881.
  20. ↑ Chen, Xiaoyou; Wang, Bin; Yang, Wen; Kong, Fanrong; Li, Chuanyou; Sun, Zhaogang; Jelfs, Peter; Gilbert, Gwendolyn L. (2014-05-01). "Rolling Circle Amplification for Direct Detection of rpoB Gene Mutations in Mycobacterium tuberculosis Isolates from Clinical Specimens". Journal of Clinical Microbiology. 52 (5): 1540–1548. doi:10.1128/JCM.00065-14. ISSN   0095-1137. PMC   3993705 . PMID   24574296.
  21. ↑ Liu, Yang; Guo, Yan-Ling; Jiang, Guang-Lu; Zhou, Shi-Jie; Sun, Qi; Chen, Xi; Chang, Xiu-Jun; Xing, Ai-Ying; Du, Feng-Jiao (2013-06-04). "Application of Hyperbranched Rolling Circle Amplification for Direct Detection of Mycobacterium Tuberculosis in Clinical Sputum Specimens". PLOS ONE. 8 (6): e64583. Bibcode:2013PLoSO...864583L. doi: 10.1371/journal.pone.0064583 . ISSN   1932-6203. PMC   3672175 . PMID   23750210.
  22. ↑ Guo, Maoxiang; Hernández-Neuta, Iván; Madaboosi, Narayanan; Nilsson, Mats; Wijngaart, Wouter van der (2018-02-12). "Efficient DNA-assisted synthesis of trans-membrane gold nanowires". Microsystems & Nanoengineering. 4: 17084. doi: 10.1038/micronano.2017.84 . ISSN   2055-7434.
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