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Rolling-circle amplification (RCA)

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Also listed as: RCA
Related terms
Background
Methods
Research
Implications
Limitations
Safety
Future research
Author information
Bibliography

Related Terms
  • Adenine, bacteria, biometrics, circular probes, cytosine, deoxyribonucleic acid, DNA polymerases, DNA/RNA diagnostics, forensics, fungi, guanine, Human Genome Project, isothermic, malaria, mutation, parasites, pathogens, PCR, pharmacogenomics, proteomics, polymerase chain reaction, RCA, thymine, viruses.

Background
  • Rolling-circle amplification (RCA) is a method for detecting specific molecules within a cell. It can identify deoxyribonucleic acid (DNA), ribonucleic acid (RNA), and proteins. When a specific molecule is present in only one type of cell, RCA can identify that type of cell. RCA is therefore a way to identify an infecting pathogen (germ), a mutation in a cancer cell, the reason one person responds to a certain drug while another does not, and limitless other applications.
  • DNA is a molecule found within the nucleus of every cell of every living organism. It contains the blueprints for that organism's growth and function. DNA is a very long and complex chemical that contains small chemicals called nucleotides, represented by four letters in a coded sequence: adenine, A; thymine, T; guanine, G; and cytosine, C. Consequently, the code reads "AATGCGCCTTTGAGGTC" and so on. A gene is a short segment of DNA that can be assigned a function, such as designating the structure of a protein.
  • Three letters designate an amino acid. Amino acids arranged linearly form a protein. Proteins are the workers in the body; they do all the construction, make up a good portion of what is built, and perform most of the operations required for the activities of life. There are roughly 30,000 proteins in a human body. Some proteins make the other nonprotein chemicals such as cortisone and adrenaline that the body needs to function. This process creates and sustains life in all its forms, and all of these chemicals can now be identified and characterized in a multi-step process that begins with RCA.

Methods
  • Specimens to be analyzed for their molecular content are often exceedingly small. These specimens, which usually contain only a few molecules, are much too small to be tested. Consequently, they must be enlarged by one of several processes called amplification. Rolling-circle amplification (RCA) is one of these methods.
  • RCA begins by generating a "probe" from a known molecule. The probe is a complementary molecule that will bind only with that known molecule. The probe is added to the specimen to be identified after the DNA in the specimen has been stabilized, or prevented from operating on its own. If the DNA were operating on its own, it would produce extraneous molecules that would interfere with the amplification process. The probe creates a circular template that can be copied many times over. During the copying process, a label is attached to the product. This label is radioactive, meaning that it will glow under ultraviolet light or will emit light when treated with certain chemicals so that it can be seen under a microscope. Only the target molecule with the probe attached can then be seen. The same probing process can be used to identify RNA and proteins.

Research
  • General: Rolling-circle amplification (RCA) can be used, along with the other related techniques, to uncover the molecular basis of life. As such it has nearly infinite potential for curing disease, enhancing food and other natural resources, improving the environment, and identifying people.
  • The most prominent area of research using RCA is the identification of specific pathogens (germs) in humans, animals, and even plants by recognizing unique DNA sequences. All kinds of germs, including viruses, bacteria, fungi, and parasites, can now be identified in a matter of hours instead of the days or weeks required by standard culturing techniques. In addition, samples too small to be identified by previous methods can be identified using RCA.
  • Cancer treatment: Cancer is an active area of research using RCA. Present efforts are directed primarily at building a database of genes. Once a vast amount of data are accumulated, that data may be analyzed until it becomes clear what targets are necessary for the survival of a particular cancer. By identifying these targets researchers can identify means of inactivating them.
  • For example, the sequence of events that helps blood vessels grow into a cancerous tumor is necessary to provide oxygen and nutrients to the cancer. If researchers are able to isolate a vulnerable molecule required for that sequence of events and create a means to inactivate that molecule, further growth of the cancer may be stopped. This process can be tested in cell cultures, then laboratory animals, then healthy human volunteers, and then cancer patients to see if it is effective and safe.
  • The current state of cancer research allows only a one-at-a-time approach to the many cancer-causing mutations because the currently available commercial tests identify only single molecules and mutations. This approach has yet to yield dramatic improvements in health because cancer requires many mutations to sustain itself, grow, and spread. The new techniques of molecular biology such as RCA can identify all the mutations in a cancer. This approach will ultimately lead to combined treatments that are specific to each individual and that address all the abnormal functions of a cancer. The result is known as "personalized medicine" and includes treating each individual differently based on their unique genetic characteristics that affect response to disease agents and treatment. RCA can help identify the various ways in which individuals are unique.
  • Disease susceptibility: Research is being done on infectious agents (germs) to characterize their life processes so that methods can be developed to interrupt them. This process is very similar to cancer research because infectious agents behave in many ways like cancers. Each has its own ways of surviving and injuring its host and each way is determined by its DNA. Identifying the unique DNA that causes these cancers and germs to do harm is a step toward limiting the damage. For example, breast cancers that respond to estrogen will be inhibited by estrogen blockers, and germs that do not produce penicillinase will be killed by penicillin. RCA can detect these individual characteristics.
  • Other diseases are also influenced by the genetic makeup of the individual patient. It is an individual's unique genetic structure, combined with environmental influences, that determines to a great extent whether one individual has asthma, another has heart disease, and another has arthritis. The ability of medical science to predict which diseases a person is most likely to have is not far in the future. From there, a greater understanding of how to prevent and even cure the disease will develop.
  • Drug response: Likewise, a person's individual response to drugs depends to a great extent on individual genetic variations. The science is called pharmacogenomics. An individual's genetic uniqueness will eventually predict which drugs will work and which drugs will cause adverse reactions. This will entail the development of an enormous database, years of research and high-powered computer analysis, and more years of testing on live subjects.
  • Forensics: The science of biometrics, which refers to identifying individuals by their anatomic and physiologic characteristics such as fingerprints and retinal scans, is now being used in criminal investigations and paternity cases. For example, RCA is able to identify people by their own DNA or fathers by their children's DNA.

Implications
  • Genomics techniques, including rolling-circle amplification (RCA), polymerase chain reaction (PCR), microarray analysis, and many other interrelated techniques, are in the early stages and offer many possibilities. At the present time, the vast majority of work is directed toward accumulating databases. Already there are enormous libraries of DNA sequences from humans, animals, plants, and germs, and much more is required before attention turns to applications of the accumulated data. It is the individual differences among species, particularly humans, that determine their unique identities and disposition to illnesses. From the libraries of data, individual variations may be identified, their role in health and disease determined, and the means to alter them devised. Already, preliminary results are appearing, such as targeted molecular agents to treat cancer and more rapid and sensitive means of detecting infections.
  • The goal of this research is to produce simple and accurate results using minimal time, sample sizes, and cost. RCA is a step along this path beyond PCR, which is an earlier method for amplifying small samples of molecules. PCR requires precise temperature changes during the procedure, more elaborate equipment to control the reactions, and a limited number of available reagents that are stable at extreme temperatures. RCA requires a smaller specimen, less expensive reagents, and no temperature changes during the reaction.

Limitations
  • All molecular biology techniques have strengths and weaknesses, not the least of which is that they are prone to human error. For this reason, researchers are continually seeking simpler, easier, less error-prone methods that are also less expensive.

Safety




Future research
  • Research in molecular biology continually aims for greater accuracy, more simplicity, less chance of error, reduced cost, and generalized use by anyone with a need or an interest. At the same time, unraveling the chemistry of life offers nearly infinite potential for advances in medicine, agriculture, and environmental science.
  • Future prospects include detailed knowledge of how cancers develop, grow, and spread, leading to more effective treatments; rapid identification of all manner of infecting organisms, allowing immediate effective treatment; and characterization of individual molecules responsible for allergic reactions, so they can be inhibited by specially designed drugs.

Author information
  • This information has been edited and peer-reviewed by contributors to the Natural Standard Research Collaboration (www.naturalstandard.com).

Bibliography
  1. Cheng Y, Li Z, Du B, et al. Homogeneous and label-free bioluminescence detection of single nucleotide polymorphism with rolling circle amplification. Analyst. 2008 Jun;133(6):750-2. PMID:
  2. Cheng Y, Li Z, Zhang X, et al. Homogeneous and label-free fluorescence detection of single-nucleotide polymorphism using target-primed branched rolling circle amplification. Anal Biochem. 2008 Jul 15;378(2):123-6.
  3. Genetics Home Reference. .
  4. Fujii R, Kitaoka M, Hayashi K. Error-prone rolling circle amplification: the simplest random mutagenesis protocol. Nat Protoc. 2006;1(5):2493-7.
  5. Huang G, Yin Y, Zhang L, et al. [Detection system for Xanthomonas axonopodis pv. citri using rolling circle amplification] Wei Sheng Wu Xue Bao. 2008 Mar 4;48(3):375-9.
  6. Jonstrup SP, Koch J, Kjems J. A microRNA detection system based on padlock probes and rolling circle amplification. RNA. 2006 Sep;12(9):1747-52.
  7. Kingsmore SF, Patel DD. Multiplexed protein profiling on antibody-based microarrays by rolling circle amplification. Curr Opin Biotechnol. 2003 Feb;14(1):74-81.
  8. Li J, Young CS, Lizardi PM, et al. In situ detection of specific DNA double strand breaks using rolling circle amplification. Cell Cycle. 2005 Dec;4(12):1767-73.
  9. McCarthy EL, Bickerstaff LE, da Cunha MP, et al. Nucleic acid sensing by regenerable surface-associated isothermal rolling circle amplification. Biosens Bioelectron. 2007 Feb 15;22(7):1236-44.
  10. National Human Genome Research Institute (NHGRI). .
  11. Natural Standard: The Authority on Integrative Medicine. .
  12. Nilsson M. Lock and roll: single-molecule genotyping in situ using padlock probes and rolling-circle amplification. Histochem Cell Biol. 2006 Aug;126(2):159-64.
  13. Rector A, Tachezy R, Van Ranst M. A sequence-independent strategy for detection and cloning of circular DNA virus genomes by using multiply primed rolling-circle amplification. J Virol. 2004 May;78(10):4993-8.
  14. Smolina I, Lee C, Frank-Kamenetskii M. Detection of low-copy-number genomic DNA sequences in individual bacterial cells by using peptide nucleic acid-assisted rolling-circle amplification and fluorescence in situ hybridization. Appl Environ Microbiol. 2007 Apr;73(7):2324-8.
  15. Yang L, Fung CW, Cho EJ, et al. Real-time rolling circle amplification for protein detection. Anal Chem. 2007 May 1;79(9):3320-9.

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The information in this monograph is intended for informational purposes only, and is meant to help users better understand health concerns. Information is based on review of scientific research data, historical practice patterns, and clinical experience. This information should not be interpreted as specific medical advice. Users should consult with a qualified healthcare provider for specific questions regarding therapies, diagnosis and/or health conditions, prior to making therapeutic decisions.

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