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Encyclopedia > Polymerase chain reaction
A strip of eight PCR tubes, each tube contains a 100μl reaction.

The polymerase chain reaction (PCR) is a biochemistry and molecular biology technique[1] for exponentially amplifying a fragment of DNA, via enzymatic replication, without using a living organism (such as E. coli or yeast). PCR can be used for amplification of a single or few copies of a piece of DNA across several orders of magnitude, generating millions or more copies of the DNA piece. As PCR is an in vitro technique, it can be performed without restrictions on the form of DNA, and it can be extensively modified to perform a wide array of genetic manipulations. PCR can refer to: the Communist Party of Réunion Passive Covert Radar, a radar system exploiting broadcast transmitters Passport Carrier Release, the name of the operating system that runs on Nortel Multiservice switches in carrier configurations Polymerase chain reaction Preventive cyclic retransmission method with forced retransmission onset level Program... Image File history File links Size of this preview: 731 × 599 pixelsFull resolution (800 × 656 pixel, file size: 668 KB, MIME type: image/png) File historyClick on a date/time to view the file as it appeared at that time. ... Image File history File links Size of this preview: 731 × 599 pixelsFull resolution (800 × 656 pixel, file size: 668 KB, MIME type: image/png) File historyClick on a date/time to view the file as it appeared at that time. ... Biochemistry is the study of the chemical processes and transformations in living organisms. ... Molecular biology is the study of biology at a molecular level. ... In mathematics, exponential growth (or geometric growth) occurs when the growth rate of a function is always proportional to the functions current size. ... The structure of part of a DNA double helix Deoxyribonucleic acid, or DNA, is a nucleic acid molecule that contains the genetic instructions used in the development and functioning of all known living organisms. ... Ribbon diagram of the enzyme TIM, surrounded by the space-filling model of the protein. ... It has been suggested that DNA replicate, Replisome, Replication fork, Lagging strand, Leading strand be merged into this article or section. ... “Life on Earth” redirects here. ... Binomial name Escherichia coli T. Escherich, 1885 Escherichia coli (usually abbreviated to E. coli) is one of the main species of bacteria that live in the lower intestines of warm-blooded animals (including birds and mammals) and are necessary for the proper digestion of food. ... Typical divisions Ascomycota (sac fungi) Saccharomycotina (true yeasts) Taphrinomycotina Schizosaccharomycetes (fission yeasts) Basidiomycota (club fungi) Urediniomycetes Sporidiales Yeasts are a growth form of eukaryotic microorganisms classified in the kingdom Fungi, with approximately 1,500 species described. ... Wiktionary has a definition of: In vitro In vitro (Latin: within glass) means within a test tube, or, more generally, outside a living organism or cell. ... An iconic image of genetic engineering; this autoluminograph from 1986 of a glowing transgenic tobacco plant bearing the luciferase gene, illustrating the possibilities of genetic engineering. ...


Developed in 1983 by Kary Mullis, PCR is now a common technique used in medical and biological research labs for a variety of tasks, such as the sequencing of genes and the diagnosis of hereditary diseases, the identification of genetic fingerprints (used in forensics and paternity testing), the detection and diagnosis of infectious diseases, and the creation of transgenic organisms. Mullis, who won the Nobel Prize for his work on PCR, credits the psychedelic drug LSD for his invention of the technique. [1](Video) Kary Banks Mullis, Ph. ... The term DNA sequencing encompasses biochemical methods for determining the order of the nucleotide bases, adenine, guanine, cytosine, and thymine, in a DNA oligonucleotide. ... A genetic disorder, or genetic disease is a disease caused, at least in part, by the genes of the person with the disease. ... Genetic fingerprinting or DNA testing is a technique to distinguish between individuals of the same species using only samples of their DNA. Its invention by Sir Alec Jeffreys at the University of Leicester was announced in 1985. ... A paternity test is conducted to prove paternity, that is, whether a man is the biological father of another individual. ... This false-colored electron micrograph shows a malaria sporozoite migrating through the midgut epithelia. ... A transgenic organism is one whose genome has been subject to artificial modification. ... The Nobel Prizes (Swedish: ) are awarded for Physics, Chemistry, Literature, Peace, and Physiology or Medicine. ... For psychedelics, see psychedelic drug. ... Lysergic acid diethylamide, commonly called LSD, LSD-25, or acid. ...

Contents

PCR principle and procedure

Figure 1: A thermal cycler for PCR

PCR is used to amplify specific regions of a DNA strand. This can be a single gene, just a part of a gene, or a non-coding sequence. Most PCR methods typically amplify DNA fragments of up to 10 kilo base pairs (kb), although some techniques allow for amplification of fragments up to 40 kb in size.[2] Photo made by Magnus Manske, hereby in the public domain This image has been released into the public domain by the copyright holder, its copyright has expired, or it is ineligible for copyright. ... Photo made by Magnus Manske, hereby in the public domain This image has been released into the public domain by the copyright holder, its copyright has expired, or it is ineligible for copyright. ... kilo base pairs (Kbp) : A thousand base pairs. ...


PCR, as currently practiced, requires several basic components [3]. These components are:

  • DNA template that contains the region of the DNA fragment to be amplified
  • One or more primers, which are complementary to the DNA regions at the 5' and 3' ends of the DNA region that is to be amplified.
  • a DNA polymerase (e.g. Taq polymerase or another DNA polymerase with a temperature optimum at around 70°C), used to synthesize a DNA copy of the region to be amplified
  • Deoxynucleotide triphosphates, (dNTPs) from which the DNA polymerase builds the new DNA
  • Buffer solution, which provides a suitable chemical environment for optimum activity and stability of the DNA polymerase
  • Divalent cations, magnesium or manganese ions; generally Mg2+ is used, but Mn2+ can be utilized for PCR-mediated DNA mutagenesis, as higher Mn2+ concentration increases the error rate during DNA synthesis [4]
  • Monovalent cation potassium ions

The PCR is carried out in small reaction tubes (0.2-0.5 ml volumes), containing a reaction volume typically of 15-100 μl, that are inserted into a thermal cycler. This is a machine that heats and cools the reaction tubes within it to the precise temperature required for each step of the reaction. Most thermal cyclers have heated lids to prevent condensation on the inside of the reaction tube caps. Alternatively, a layer of oil may be placed on the reaction mixture to prevent evaporation. A primer is a nucleic acid strand, or a related molecule that serves as a starting point for DNA replication. ... 3D structure of the DNA-binding helix-hairpin-helix motifs in human DNA polymerase beta A DNA polymerase is an enzyme that assists in DNA replication. ... Taq polymerase (Taq Pol, or simply Taq) is a thermostable polymerase used in polymerase chain reaction to check for the presence or absence of a gene by amplifying a DNA fragment. ... A nucleotide is a chemical compound that consists of a heterocyclic base, a sugar, and one or more phosphate groups. ... Acids and bases: Acid-base reaction theories pH Self-ionization of water Buffer solutions Systematic naming Electrochemistry Acid-base extraction Acids: Strong acids Weak acids Superacids Lewis acids Mineral acids Organic acids Bases: Strong bases Weak bases Superbases Lewis bases Organic bases edit Buffer solutions are solutions which resist change... In chemistry, divalent anions are atoms or radicals with 2 additional electrons when compared to their elemental state (that is, with 2 more electrons than protons); for instance, S-- is the sulfide anion. ... An ion is an atom or group of atoms with a net electric charge. ... General Name, symbol, number magnesium, Mg, 12 Chemical series alkaline earth metals Group, period, block 2, 3, s Appearance silvery white solid at room temp Standard atomic weight 24. ... General Name, symbol, number manganese, Mn, 25 Chemical series transition metals Group, period, block 7, 4, d Appearance silvery metallic Standard atomic weight 54. ... General Name, symbol, number potassium, K, 19 Chemical series alkali metals Group, period, block 1, 4, s Appearance silvery white Standard atomic weight 39. ... Thermal cycler Thermal cycler or thermocycler or PCR machine is a laboratory apparatus used for PCR. The device has a thermal block with holes where tubes with the PCR reaction mixtures can be inserted. ...


Procedure

Figure 2: Schematic drawing of the PCR cycle. (1) Denaturing at 94-96°C. (2) Annealing at ~65°C (3) Elongation at 72°C. Four cycles are shown here.

The PCR usually consists of a series of 20 to 35 cycles. Most commonly, PCR is carried out in three steps (Fig. 2), often preceded by one temperature hold at the start and followed by one hold at the end. Image File history File links No higher resolution available. ... Image File history File links No higher resolution available. ...

  1. Prior to the first cycle, during an initialization step, the PCR reaction is often heated to a temperature of 94-96°C (or 98°C if extremely thermostable polymerases are used), and this temperature is then held for 1-9 minutes. This first hold is employed to ensure that most of the DNA template and primers are denatured, i.e., that the DNA is melted by disrupting the hydrogen bonds between complementary bases of the DNA strands, yielding two single strands of DNA. Also, some PCR polymerases require this step for activation (see hot-start PCR).[5] Following this hold, cycling begins, with one step at 94-98°C for 20-30 seconds (denaturation step).
  2. The denaturation is followed by the annealing step. In this step the reaction temperature is lowered so that the primers can anneal to the single-stranded DNA template. Brownian motion causes the primers to move around, and DNA-DNA hydrogen bonds are constantly formed and broken between primer and template. Stable bonds are only formed when the primer sequence very closely matches the template sequence, and to this short section of double-stranded DNA the polymerase attaches and begins DNA synthesis. The temperature at this step depends on the melting temperature of the primers, and is usually between 50-64°C for 20-40 seconds.
  3. The annealing step is followed by an extension/elongation step during which the DNA polymerase synthesizes new DNA strands complementary to the DNA template strands. The temperature at this step depends on the DNA polymerase used. Taq polymerase has a temperature optimum of 70-74°C; thus, in most cases a temperature of 72°C is used. The hydrogen bonds between the extended primer and the DNA template are now strong enough to withstand forces breaking these attractions at the higher temperature. Primers that have annealed to DNA regions with mismatching bases dissociate from the template and are not extended. The DNA polymerase condenses the 5'-phosphate group of the dNTPs with the 3'-hydroxyl group at the end of the nascent (extending) DNA strand, i.e., the polymerase adds dNTP's that are complementary to the template in 5' to 3' direction, thus reading the template in 3' to 5' direction. The extension time depends both on the DNA polymerase used and on the length of the DNA fragment to be amplified. As a rule-of-thumb, at its optimum temperature, the DNA polymerase will polymerize a thousand bases in one minute. A final elongation step of 5-15 minutes (depending on the length of the DNA template) after the last cycle may be used to ensure that any remaining single-stranded DNA is fully extended. A final hold of 4-15°C for an indefinite time may be employed for short-term storage of the reaction, e.g., if reactions are run overnight.
Figure 3: Ethidium bromide-stained PCR products after Gel electrophoresis. Two sets of primers were used to amplify the IGF gene from three different DNA samples. In sample #1 the gene was not amplified by PCR, whereas bands for tissue #2 and #3 indicate successful amplification of the IGF gene. A positive control, and a DNA ladder containing DNA fragments of defined length (last lane to the right) to estimate fragment sizes in the experimental PCRs, were also ran on this gel

To check whether the PCR generated the anticipated DNA fragment (also sometimes referred to as amplimer), agarose gel electrophoresis is commonly employed for size separation of the PCR products. The size(s) of PCR products is thereby determined by comparison with a DNA ladder, which contains DNA fragments of known size, ran on the gel alongside the PCR products (see Fig. 3). “PCR” redirects here. ... Irreversible egg protein denaturation and loss of solubility, caused by the high temperature (while cooking it) Denaturation is the alteration of a protein or nucleic acids shape through some form of external stress (for example, by applying heat, acid or alkali), in such a way that it will no... Annealing, in genetics, means for DNA or RNA to pair by hydrogen bonds to a complementary sequence, forming a double-stranded polynucleotide. ... Three different views of Brownian motion, with 32 steps, 256 steps, and 2048 steps denoted by progressively lighter colors. ... DNA melting, also called DNA denaturation, is the process by which double-stranded deoxyribonucleic acid unwinds and separates into single-stranded strands through the breaking of hydrogen bonding between the bases. ... Taq polymerase (Taq Pol, or simply Taq) is a thermostable polymerase used in polymerase chain reaction to check for the presence or absence of a gene by amplifying a DNA fragment. ... In chemistry, a phosphate is a polyatomic ion or radical consisting of one phosphorus atom and four oxygen. ... A nucleotide is a chemical compound that consists of a heterocyclic base, a sugar, and one or more phosphate groups. ... Hydroxide is a functional group consisting of oxygen and hydrogen: -O−H It has a charge of 1-. The term hydroxyl group is used when the functional group -OH is counted as a substituent of an organic compound. ... Image File history File links Roland_Gel. ... Image File history File links Roland_Gel. ... Gel electrophoresis is the separation of deoxyribonucleic acid, ribonucleic acid, or protein through an electric charge. ... Look up primer in Wiktionary, the free dictionary. ... For other uses, see Gene (disambiguation). ... Digital image of 3 plasmid restriction digests run on a 1% w/v agarose gel, 3 Volts/cm, stained with ethidium bromide. ...


PCR optimization

Main article: PCR optimization

In practice, PCR can fail for various reasons, in part due to its sensitivity to contamination causing amplification of spurious DNA products. Because of this, a number of techniques and procedures have been developed for optimizing PCR conditions. Contamination with extraneous DNA is addressed with lab protocols and procedures that separate pre-PCR reactions from potential DNA contaminants.[3] This usually involves spatial separation of PCR-setup areas from areas for analysis or purification of PCR products, and thoroughly cleaning the work surface between reaction setups. Primer-design techniques are important in improving PCR product yield and in avoiding the formation of spurious products, and the usage of alternate buffer components or polymerase enzymes can help with amplification of long or otherwise problematic regions of DNA. The polymerase chain reaction (PCR) is a commonly used molecular biology tool for amplifying DNA, and various techniques for PCR optimization have been developed by molecular biologists to improve PCR performance and minimize failure. ...


Uses of PCR

Isolation of genomic material

Because it selectively amplifies a specific region of DNA, PCR can be used to isolate desired sections of DNA sequence from whole genomic material. Many techniques (such as Southern and northern blotting and DNA cloning) require a pool of DNA molecules isolated from a particular DNA region — although PCR is not always a necessary prerequisite, its usage has greatly augmented these methods. Because PCR also amplifies the isolated region, the techniques are endowed with high amounts of pure starting material and become applicable to samples otherwise too small for analysis. A Southern blot is a method routinely used in molecular biology to check for the presence of a DNA sequence in a DNA sample. ... The northern blot is a technique used in molecular biology research to study gene expression. ... Cloning is the process of creating an identical copy of an original. ...

Sequencing and the detection of genetic diseases

Once a sample is created where all molecules derive from a single region of DNA, it is possible to go forward with DNA sequencing to determine the unknown sequence of nucleotides that spans the fragment between the two primers. One of the PCR primers is commonly used as the anchor for Sanger sequencing, which is currently the most common method for DNA sequencing. This method is commonly used to diagnose genetic disorders; a doctor can confirm a diagnosis by observing DNA sequence differences that are known to be associated with a disorder. The term DNA sequencing encompasses biochemical methods for determining the order of the nucleotide bases, adenine, guanine, cytosine, and thymine, in a DNA oligonucleotide. ...

Cloning a gene using a plasmid.
(1) Chromosomal DNA of organism A. (2) PCR. (3) Multiple copies of a single gene from organism A. (4) Insertion of the gene into a plasmid. (5) Plasmid with gene from organism A. (6) Insertion of the plasmid in organism B. (7) Multiplication or expression of the gene, originally from organism A, occurring in organism B.
Recombinant DNA techniques

Isolating a DNA sequence also enables recombinant DNA technologies, which involve the insertion of a given DNA sequence into the genetic material of another organism (now termed a genetically modified organism (GMO)). PCR is often used to amplify a gene, which can then be inserted into a target genome through homologous recombination or, more commonly, inserted into a vector that carries the DNA into the target organism, such as a plasmid. From Nupedia File links The following pages link to this file: Polymerase chain reaction Categories: GFDL images ... From Nupedia File links The following pages link to this file: Polymerase chain reaction Categories: GFDL images ... Recombinant DNA (rDNA) is an artificial DNA sequence resulting from the combination of different DNA sequences. ... A genetically modified organism (GMO) is an organism whose genetic material has been altered using the genetic engineering techniques generally known as recombinant DNA technology. ... Chromosomal crossover is the process by which two chromosomes, paired up during Prophase I of meiosis, exchange some distal portion of their DNA. Crossover occurs when two chromosomes, normally two homologous instances of the same chromosome, break and then reconnect but to the different end piece. ... In epidemiology, a vector is an organism that does not cause disease itself but which spreads infection by conveying pathogens from one host to another. ... Figure 1: Illustration of a bacterium with plasmids enclosed showing chromosomal DNA and plasmids. ...

Genetic fingerprinting and paternity testing

Genetic fingerprinting is a forensic technique used to identify a person by comparing his or her DNA with the DNA in a given sample. PCR can be used to amplify a specific set regions which vary in length from person to person. The combination of all these variations creates "genetic fingerprint" – when these fragments are run out on an agarose gel they produce a specific pattern of bands. With the use of PCR, in theory, only a single DNA strand is needed, providing very high sensitivity to this technique, although such sensitive amplification increases the risk of confounding results due to possible contamination with, and amplification of, DNA from extraneous sources. There are different PCR-based methods for fingerprinting, summarized in Genetic fingerprinting. The overall pattern of PCR-generated DNA fragments after gel electrophoresis and visualization by ethidium bromide staining (or hybridization with a DNA probe after Southern blotting), can be considered a DNA fingerprint analogous to the fingerprint pattern unique to each individual. Genetic fingerprinting, DNA testing, DNA typing, and DNA profiling are techniques used to distinguish between individuals of the same species using only samples of their DNA. Its invention by Sir Alec Jeffreys at the University of Leicester was announced in 1985. ... A polysaccharide obtained from agar that is the most widely used medium for gel electrophoresis procedures. ... Genetic fingerprinting, DNA testing, DNA typing, and DNA profiling are techniques used to distinguish between individuals of the same species using only samples of their DNA. Its invention by Sir Alec Jeffreys at the University of Leicester was announced in 1985. ... Gel electrophoresis is the separation of deoxyribonucleic acid, ribonucleic acid, or protein through an electric charge. ... R-phrases , S-phrases , , , , , Flash point > 100 °C Except where noted otherwise, data are given for materials in their standard state (at 25 Â°C, 100 kPa) Infobox disclaimer and references Absorption spectrum of ethidium bromide Ethidium bromide (sometimes abbreviated as EtBr) is an intercalating agent commonly used as a nucleic... A hybridization probe is a short piece of DNA (on the order of 100-500 bases) that is denatured (by heating) into single strands and then radioactively labeled, usually with phosphorus (32P or 33P). ... A Southern blot is a method routinely used in molecular biology to check for the presence of a DNA sequence in a DNA sample. ... A macro shot of a palm and the base of several fingers; as seen here, debris can gather between the ridges. ...

Figure 4: Electrophoresis of PCR-amplified DNA fragments. (1) Father. (2) Child. (3) Mother. The child has inherited some, but not all of the fingerprint of each of its parents, giving it a new, unique fingerprint.

Although these resulting 'fingerprints' are unique , genetic relationships, for example, parent-child or siblings, can be determined from two or more genetic fingerprints, which can be used for paternity tests (Fig. 4). A variation of this technique can also be used to determine evolutionary relationships between organisms. From Nupedia File links The following pages link to this file: Polymerase chain reaction Categories: GFDL images ... From Nupedia File links The following pages link to this file: Polymerase chain reaction Categories: GFDL images ...


Amplification of DNA & DNA quantitation

Because PCR amplifies the regions of DNA that it targets, PCR can be used to analyze extremely small amounts of sample. This is often critical for forensic analysis, when only a trace amount of DNA is available as evidence. The amount of time it takes to amplify DNA to a given level is related to the original amount in the sample, which means that PCR can also be used to quantify the presence of particular DNA fragments.

Analysis of ancient DNA

Using PCR, it becomes possible to analyze DNA that is thousands of years old. this technique also helps us to do dating of bones. PCR techniques have been successfully used on animals, such as a forty-thousand-year-old mammoth, and also on human DNA, in applications ranging from the analysis of Egyptian mummies to the identification of a Russian Tsar[6]. This article is about the genus Mammuthus. ... A mummy is a corpse whose skin and dried flesh have been preserved by either intentional or accidental exposure to chemicals, extreme cold, very low humidity, or airlessness. ... Tsar (Bulgarian, Serbian and Macedonian цар, Russian  , in scientific transliteration respectively car and car ), occasionally spelled Czar or Tzar and sometimes Csar or Zar in English, is a Slavonic term designating certain monarchs. ...

Detection of viral DNA

Viral DNA can likewise be detected by PCR amplification. Primers need to be specific to sequences in the DNA of a given virus, and the PCR can be used for diagnostic analyses or DNA sequencing of parts or all of the viral genome. Because of the high sensitivity of PCR, virus detection may be possible soon after infection and even before the onset of disease symptoms. Such early detection may give physicians a significant lead in treatment. The the amount of virus ("viral load") in a patient can be quantified by PCR-based DNA quantitation techniques (see below). This article is about biological infectious particles. ...

DNA quantitation and comparison of gene expression

Because the amount of product produced by PCR roughly correlates to the amount of starting material, PCR can be used to estimate the amount of a given sequence that is present in a sample – a technique especially useful for determining gene expression levels. In cells, each gene is expressed through the production of messenger RNA (mRNA), which is then used to create a protein corresponding to the gene. The amount of mRNA in the cell for a given gene reflects how active that gene is. By using reverse transcription to produce DNA complementary to the mRNA (called cDNA) and subsequently using PCR to amplify these molecules, the amount of DNA produced for each gives a rough measure of the underlying expression for that gene. Reverse transcriptase is an enzyme used by all retroviruses and retrotransposons that transcribes the genetic information from the virus or retrotransposon from RNA into DNA, which can integrate into the host genome. ...


More accurate measurements are possible when the amount of double-stranded DNA is measured after each round of PCR amplification, a technique known as "quantitative real-time PCR". A dye that fluoresces in the presence of double-stranded DNA is added to the mixture and, as the fluorescence increases, the timing as well as amount of amplification can be measured.


History

A 1971 paper in the Journal of Molecular Biology by Kleppe and co-workers first described a method using an enzymatic assay to replicate a short DNA template with primers in vitro. [7] However, this early manifestation of the basic PCR principle did not receive much attention, and the invention of the polymerase chain reaction in 1983 is generally credited to Kary Mullis [8][9]. He was awarded the Nobel Prize in Chemistry in 1993 for his invention, seven years after he and his colleagues at Cetus first put his proposal to practice. However, some controversies have remained about the intellectual and practical contributions of other scientists to Mullis' work, and whether he had been the sole inventor of the PCR principle. (see main article:Kary Mullis) Published weekly by Elsevier, under the Academic Press imprint, the Journal of Molecular Biology (ISSN: 0022-2836) publishes original scientific research concerning studies of organisms or their components at the molecular level. ... Kary Banks Mullis, Ph. ... This is a list of Nobel Prize laureates in Chemistry from 1901 to 2006. ... Year 1993 (MCMXCIII) was a common year starting on Friday (link will display full 1993 Gregorian calendar). ... Kary Banks Mullis, Ph. ...


At the time he developed PCR in 1983, Mullis was working in Emeryville, California for Cetus Corporation, one of the first biotechnology companies. There, he was responsible for synthesizing short chains of DNA. Mullis has written that he conceived of PCR while cruising along the Pacific Coast Highway one night in his car[8]. He was playing in his mind with a new way of analyzing changes (mutations) in DNA when he realized that he had instead invented a method of amplifying any DNA region through repeated cycles of duplication driven by an enzyme called DNA polymerase. Emeryville is a small city located in Alameda County, California. ... Cetus was a biotechnology company established in Berkeley, California in 1972. ... The structure of insulin Biological technology is technology based on biology, especially when used in agriculture, food science, and medicine. ... Scene from highway 1 (Near Half Moon Bay) State Route 1, often called Highway 1, is a state highway that runs along a large length of the Pacific coast of the U.S. State of California. ... Ribbon diagram of the enzyme TIM, surrounded by the space-filling model of the protein. ... 3D structure of the DNA-binding helix-hairpin-helix motifs in human DNA polymerase beta A DNA polymerase is an enzyme that assists in DNA replication. ...


In Scientific American, Mullis summarized the accomplishment: "Beginning with a single molecule of the genetic material DNA, the PCR can generate 100 billion similar molecules in an afternoon. The reaction is easy to execute. It requires no more than a test tube, a few simple reagents, and a source of heat."[10] Scientific American is a popular-science magazine, published (first weekly and later monthly) since August 28, 1845, making it the oldest continuously published magazine in the United States. ...


DNA polymerase occurs naturally in living organisms. In cells it functions to duplicate DNA when cells divide in mitosis and meiosis. Polymerase works by binding to a single DNA strand and creating the complementary strand. In the first of many original processes, the enzyme was used in vitro (in a controlled environment outside an organism). The double-stranded DNA was separated into two single strands by heating it to 94°C (201°F). At this temperature, however, the DNA polymerase used at the time were destroyed, so the enzyme had to be replenished after the heating stage of each cycle. The original procedure was very inefficient, since it required a great deal of time, large amounts of DNA polymerase, and continual attention throughout the process. It has been suggested that Binary fission be merged into this article or section. ... Not to be confused with miosis. ... Ribbon diagram of the enzyme TIM, surrounded by the space-filling model of the protein. ... Wiktionary has a definition of: In vitro In vitro (Latin: within glass) means within a test tube, or, more generally, outside a living organism or cell. ...


In 1986, this original PCR process was greatly improved by the use of DNA polymerase taken from thermophilic bacteria grown in geysers at a temperature of over 110°C (230°F). The DNA polymerase taken from these organisms is stable at high temperatures and, when used in PCR, does not break down when the mixture was heated to separate the DNA strands. Since there was no longer a need to add new DNA polymerase for each cycle, the process of copying a given DNA strand could be simplified and automated. This article is about an organism. ... Strokkur geyser, Iceland A geyser is a type of hot spring that erupts periodically, ejecting a column of hot water and steam into the air. ...


One of the first thermostable DNA polymerases was obtained from Thermus aquaticus and was called "Taq." Taq polymerase is widely used in current PCR practice. A disadvantage of Taq is that it sometimes makes mistakes when copying DNA, leading to mutations (errors) in the DNA sequence, since it lacks 3'→5' proofreading exonuclease activity. Polymerases such as Pwo or Pfu, obtained from Archaea, have proofreading mechanisms (mechanisms that check for errors) and can significantly reduce the number of mutations that occur in the copied DNA sequence. However these enzymes polymerise DNA at a much slower rate than Taq. Combinations of both Taq and Pfu are available nowadays that provide both high processivity (fast polymerisation) and high fidelity (accurate duplication of DNA). Binomial name Thermophilus aquaticus Brock & Freeze, 1969 Thermophilus aquaticus is a species of bacterium that can tolerate high temperatures; it is the source of the heat-resistant enzyme Taq DNA Polymerase, one of the most important enzymes in molecular biology because of its use in the polymerase chain reaction. ... It has been suggested that mutant be merged into this article or section. ... Phyla Crenarchaeota Euryarchaeota Korarchaeota Nanoarchaeota ARMAN The Archaea (), or archaebacteria, are a major group of microorganisms. ...


PCR has been performed on DNA larger than 10 kilobases, but the average PCR is only several hundred to a few thousand bases of DNA. The problem with long PCR is that there is a balance between accuracy and processivity of the enzyme. Usually, the longer the fragment, the greater the probability of errors.


Patent wars

The PCR technique was patented by Cetus Corporation, where Mullis worked when he invented the technique in 1983. The Taq polymerase enzyme was also covered by patents. There have been several high-profile lawsuits related to the technique, including an unsuccessful lawsuit brought by DuPont. The pharmaceutical company Hoffmann-La Roche purchased the rights to the patents in 1992 and currently holds those that are still protected. Cetus was a biotechnology company established in Berkeley, California in 1972. ... Dupont, DuPont, Du Pont, or du Pont may refer to: // E.I. du Pont de Nemours and Company, the worlds third largest chemical company Du Pont Motors Gilbert Dupont, a French stock brokerage part of retail banking network Crédit du Nord ST Dupont, a French manufacturer of fine... Hoffmann-La Roche, Ltd. ... Year 1992 (MCMXCII) was a leap year starting on Wednesday (link will display full 1992 Gregorian calendar). ...


A related patent battle over the Taq polymerase enzyme is still ongoing in several jurisdictions around the world between Roche and Promega. Interestingly, it seems possible that the legal arguments will extend beyond the life of the original PCR and Taq polymerase patents, which expired on March 28, 2005.[11] Promega develops, commercializes, and manufactures reagents, supplies, assays and equipment for biological research, primarily serving the molecular biology and cell biology research sectors. ...


Variations on the basic PCR technique

  • Allele-specific PCR - AS-PCR is used to determine the genotype of single-nucleotide polymorphisms (SNPs) (single base differences in DNA) by using primers whose ends overlap the SNP and differ by that single base. PCR amplification is less efficient in the presence of a mismatch, so the differences in amplification resulting from different primers can be used to quickly determine which primer matches the sample genotype.
  • Assembly PCR - Assembly PCR is the completely artificial synthesis of long gene products by performing PCR on a pool of long oligonucleotides with short overlapping segments. The oligonucleotides alternate between sense and antisense directions, and the overlapping segments serve to order the PCR fragments so that they selectively produce their final product.
  • Asymmetric PCR - Asymmetric PCR is used to preferentially amplify one strand of the original DNA more than the other. It finds use in some types of sequencing and hybridization probing where having only one of the two complementary stands is required. PCR is carried out as usual, but with a great excess of the primers for the chosen strand. Due to the slow (arithmetic) amplification later in the reaction after the limiting primer has been used up, extra cycles of PCR are required. A recent modification on this process, known as Linear-After-The-Exponential-PCR (LATE-PCR), uses a limiting primer with a higher melting temperature (Tm) than the excess primer to maintain reaction efficiency as the limiting primer concentration decreases mid-reaction.
  • Colony PCR - Bacterial clones (E.coli) can be rapidly screened for correct DNA vector constructs. Selected bacterial colonies are picked with a sterile toothpick from an agarose plate and dabbed into the master mix or sterile water. Primers (and the master mix) are added, and the PCR is started with an extended time at 95˚C when standard polymerase is used or with a shortened denaturation step at 100˚C and special chimeric DNA polymerase.[12]
  • Helicase-dependent amplification - similar to traditional PCR, but maintains a constant temperature rather than cycling through denaturation and annealing/extension cycles. Helicase, an enzyme that unwinds DNA, is used in place of thermal denaturation.[13]
  • Hot-start PCR is a technique that reduces non-specific amplification during the initial set up stages of the PCR. The technique may be performed manually by simply heating the reaction components briefly at the melting temperature (e.g., 95˚C) before adding the polymerase.[14] Specialized enzyme systems have been developed that inhibit the polymerase's activity at ambient temperature, either by the binding of an antibody[5] or by the presence of covalently bound inhibitors that only dissociate after a high-temperature activation step. Hot-start/cold-finish PCR is achieved with new hybrid polymerases that are inactive at ambient temperature and are instantly activated at elongation temperature.
  • Intersequence-specific (ISSR) PCR - a PCR method for DNA fingerprinting that amplifies regions between some simple sequence repeats to produce a unique fingerprint of amplified fragment lengths.[15]
  • Inverse PCR - Inverse PCR is a method used to allow PCR when only one internal sequence is known. This is especially useful in identifying flanking sequences to various genomic inserts. This involves a series of DNA digestions and self ligation, resulting in known sequences at either end of the unknown sequence.
  • Ligation-mediated PCR-Ligation-mediated PCR (LM-PCR) can be used to detect vector insertion sites into a genome.
  • Methylation-specific PCR (MSP) - The MSP method was developed by Stephen Baylin and Jim Herman at the Johns Hopkins School of Medicine, [16] and is used to detect methylation of CpG islands in genomic DNA. DNA is first treated with sodium bisulfite, which converts unmethylated cytosine bases to uracil, which is recognized by PCR primers as thymine. Two PCR reactions are then carried out on the modified DNA, using primer sets identical except at any CpG islands within the primer sequences. At these points, one primer set recognizes DNA with cytosines to amplify methylated DNA, and one set recognizes DNA with uracil or thymine to amplify unmethylated DNA. MSP using qPCR can also be performed to obtain quantitative rather than qualitative information about methylation.
  • Multiplex Ligation-dependent Probe Amplification (MLPA) - permits multiple targets to be amplified with only a single primer pair, thus avoiding the resolution limitations of multiplex PCR (see below).
  • Multiplex-PCR - The use of multiple, unique primer sets within a single PCR reaction to produce amplicons of varying sizes specific to different DNA sequences. By targeting multiple genes at once, additional information may be gained from a single test run that otherwise would require several times the reagents and more time to perform. Annealing temperatures for each of the primer sets must be optimized to work correctly within a single reaction, and amplicon sizes, i.e., their base pair length, should be different enough to form distinct bands when visualized by gel electrophoresis.
  • Nested PCR - Nested PCR increases the specificity of DNA amplification, by reducing background due to non-specific amplification of DNA. Two sets of primers are being used in two successive PCR reactions. In the first reaction, one pair of primers is used to generate DNA products, which besides the intended target, may still consist of non-specifically amplified DNA fragments. The product(s) (sometimes after gel purification after electrophoresis of the PCR product) are then used in a second PCR reaction with a set of primers whose binding sites are completely or partially different from the primer pair used in the first reaction, but are completely within the DNA target fragment. Nested PCR is often more successful in specifically amplifying long DNA fragments than conventional PCR, but it requires more detailed knowledge of the target sequences.
  • Quantitative PCR - Q-PCR (Quantitative PCR) is used to measure the quantity of a PCR product (preferably real-time). It is the method of choice to quantitatively measure starting amounts of DNA, cDNA or RNA. Q-PCR is commonly used to determine whether a DNA sequence is present in a sample and the number of its copies in the sample. The method with currently the highest level of accuracy is Quantitative real-time PCR. It is often confusingly known as RT-PCR (Real Time PCR) or RQ-PCR. QRT-PCR or RTQ-PCR are more appropriate contractions. RT-PCR commonly refers to reverse transcription PCR (see below), which is often used in conjunction with Q-PCR. QRT-PCR methods use fluorescent dyes, such as Sybr Green, or fluorophore-containing DNA probes, such as TaqMan, to measure the amount of amplified product in real time.
  • RT-PCR - RT-PCR (Reverse Transcription PCR) is a method used to amplify, isolate or identify a known sequence from a cellular or tissue RNA. The PCR reaction is preceded by a reaction using reverse transcriptase to convert RNA to cDNA. RT-PCR is widely used in expression profiling, to determine the expression of a gene or to identify the sequence of an RNA transcript, including transcription start and termination sites and, if the genomic DNA sequence of a gene is known, to map the location of exons and introns in the gene. The 5' end of a gene (corresponding to the transcription start site) is typically identified by a RT-PCR method, named RACE-PCR, short for Rapid Amplification of cDNA Ends.
  • TAIL-PCR - thermal asymmetric interlaced PCR is used to isolate unknown sequence flanking a known sequence. Within the known sequence TAIL-PCR uses a nested pair of primers with differing annealing temperatures; a degenerate primer is used to amplify in the other direction from the unknown sequence.[17]
  • Touchdown PCR - Touchdown PCR is a variant of PCR that aims to reduce nonspecific background by gradually lowering the annealing temperature as PCR cycling progresses. The annealing temperature at the initial cycles is usually a few degrees above the Tm of the primers used, while at the later cycles, it is a few degrees below the primer Tm. The higher temperatures give greater specificity for primer binding, and the lower temperatures permit more efficient amplification from the specific products formed during the initial cycles.
  • PAN-AC - Another isothermal amplification reaction. The authors claim that it can be used in living cells.[18][19].

A Single Nucleotide Polymorphism or SNP (pronounced snip) is a DNA sequence variation, occurring when a single nucleotide: adenine (A), thymine (T), cytosine (C) or guanine (G) - in the genome is altered. ... The structure of part of a DNA double helix Deoxyribonucleic acid, or DNA, is a nucleic acid molecule that contains the genetic instructions used in the development and functioning of all known living organisms. ... In genetics and biochemistry, sequencing means to determine the primary structure (or primary sequence) of an unbranched biopolymer. ... Arithmetic tables for children, Lausanne, 1835 Arithmetic or arithmetics (from the Greek word αριθμός = number) is the oldest and most elementary branch of mathematics, used by almost everyone, for tasks ranging from simple day-to-day counting to advanced science and business calculations. ... The dissociation of a double-stranded DNA molecule is often referred to as melting because it occurs quickly once a certain temperature has been reached. ... Binomial name Escherichia coli T. Escherich, 1885 E. coli at 10,000x magnification Escherichia coli (usually abbreviated to E. coli) is one of the main species of bacteria that live in the lower intestines of warm-blooded animals (including birds and mammals) and are necessary for the proper digestion of... Figure 1: Illustration of a bacterium with plasmids enclosed showing chromosomal DNA and plasmids. ... Helicase-dependent amplification (HDA) is a method for in vitro DNA amplification like the polymerase chain reaction. ... Each antibody binds to a specific antigen; an interaction similar to a lock and key. ... Inverse PCR is a method used to allow PCR when only one internal sequence is known. ... Flank is a word which might mean any of several different things: A flank is the side of either a horse or a military unit. ... In biology the genome of an organism is the whole hereditary information of an organism that is encoded in the DNA (or, for some viruses, RNA). ... A restriction digest is a molecular biology procedure used to prepare DNA for analysis or other processing. ... Self Ligation is an orthodontic term used to describe a bracket with a sliding or rotating mechanism to ligate an archwire. ... Multiplex ligation-dependent probe amplification (MLPA) is a variation of the polymerase chain reaction that permits multiple targets to be amplified with only a single primer pair[1]. Each probe consists of a pair of primers that straddle the target site of interest and are subsequently ligated into a complete... PCR machine Amplicons are the products of polymerase chain reactions (PCR) or ligase chain reactions (LCR). ... Gel electrophoresis is the separation of deoxyribonucleic acid, ribonucleic acid, or protein through an electric charge. ... Nested PCR is a modification of PCR intended to reduce the contaminations in products due to the amplification of unexpected primer binding sites. ... Quantitative PCR (Q-PCR) is a modification of PCR used to rapidly measure the quantity of PCR product (preferably real-time), thus is an indirect method for quantitatively measuring starting amounts of DNA, cDNA or RNA. This is commonly used for the purpose of determening whether a sequence is present... Real-Time PCR, also called quantitative (real-time) PCR, is a method of simultaneous DNA quantification and amplification. ... RT-PCR (Reverse Transcription-PCR) is a modification of PCR in which an RNA strand is first reverse transcribed into its DNA complement or cDNA, followed by amplification of the resulting DNA using PCR. This can either be a 1 or 2 step process. ... A fluorophore is a component of a molecule which causes a molecule to be fluorescent. ... This article or section is in need of attention from an expert on the subject. ... RT-PCR (Reverse Transcription-PCR) is a modification of PCR in which an RNA strand is first reverse transcribed into its DNA complement or cDNA, followed by amplification of the resulting DNA using PCR. This can either be a 1 or 2 step process. ... Drawing of the structure of cork as it appeared under the microscope to Robert Hooke from Micrographia which is the origin of the word cell being used to describe the smallest unit of a living organism Cells in culture, stained for keratin (red) and DNA (green) The cell is the... Biological tissue is a collection of interconnected cells that perform a similar function within an organism. ... Ribonucleic acid or RNA is a nucleic acid polymer consisting of nucleotide monomers that plays several important roles in the processes that translate genetic information from deoxyribonucleic acid (DNA) into protein products; RNA acts as a messenger between DNA and the protein synthesis complexes known as ribosomes, forms vital portions... In biochemistry, a reverse transcriptase, also known as RNA-dependent DNA polymerase, is a DNA polymerase enzyme that transcribes single-stranded RNA into double-stranded DNA. Normal transcription involves the synthesis of RNA from DNA, hence reverse transcription is the reverse of this. ... Ribonucleic acid or RNA is a nucleic acid polymer consisting of nucleotide monomers that plays several important roles in the processes that translate genetic information from deoxyribonucleic acid (DNA) into protein products; RNA acts as a messenger between DNA and the protein synthesis complexes known as ribosomes, forms vital portions... In genetics, complementary DNA (cDNA) is single-stranded DNA synthesized from a mature mRNA template. ... Expression Profiling Microarray technology is often used for expression profiling. ... The exon portion of a DNA strand encodes a specific portion of a protein. ... Diagram of the location of introns and exons within a gene. ... RACE, 5 RACE or Rapid Amplification of cDNA Ends, is a molecular biology technique used to amplify the 5 ends of cDNA by a special PCR reaction. ... Touchdown PCR or Touchdown style PCR is a method of PCR (polymerase chain reaction) by which degenerate primers will avoid amplifying nonspecific sequence. ...

References

  1. ^ The history of PCR: Smithsonian Institution Archives, Institutional History Division. Retrieved 24 June 2006.
  2. ^ Cheng S, Fockler C, Barnes WM, Higuchi R (1994). "Effective amplification of long targets from cloned inserts and human genomic DNA". Proc Natl Acad Sci. 91: 5695-5699. PMID 8202550. 
  3. ^ a b Joseph Sambrook and David W. Russel (2001). Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory Press. ISBN 0-87969-576-5.  Chapter 8: In vitro Amplification of DNA by the Polymerase Chain Reaction
  4. ^ Pavlov AR, Pavlova NV, Kozyavkin SA, Slesarev AI (2004). "Recent developments in the optimization of thermostable DNA polymerases for efficient applications". Trends Biotechnol. 22: 253-260. PMID 15109812. 
  5. ^ a b D.J. Sharkey, E.R. Scalice, K.G. Christy Jr., S.M. Atwood, and J.L. Daiss (1994). "Antibodies as Thermolabile Switches: High Temperature Triggering for the Polymerase Chain Reaction". Bio/Technology 12: 506-509. 
  6. ^ http://photoscience.la.asu.edu/photosyn/courses/BIO_343/lecture/DNAtech.html
  7. ^ Kleppe K, Ohtsuka E, Kleppe R, Molineux I, Khorana HG (1971). "Studies on polynucleotides. XCVI. Repair replications of short synthetic DNA's as catalyzed by DNA polymerases". J. Mol. Biol. 56: 341-361. 
  8. ^ a b Mullis, Kary (1998). Dancing Naked in the Mind Field. New York: Pantheon Books. ISBN 0-679-44255-3. 
  9. ^ Rabinow, Paul (1996). Making PCR: A Story of Biotechnology. Chicago: University of Chicago Press. ISBN 0-226-70146-8. 
  10. ^ Mullis, Kary (1990). "The unusual origin of the polymerase chain reaction". Scientific American 262 (4): 56-61, 64-5. 
  11. ^ Advice on How to Survive the Taq Wars ¶2: GEN Genetic Engineering News Biobusiness Channel: Article. May 1 2006 (Vol. 26, No. 9).
  12. ^ Pavlov AR, Pavlova NV, Kozyavkin SA, Slesarev AI (2006). "Thermostable DNA Polymerases for a Wide Spectrum of Applications: Comparison of a Robust Hybrid TopoTaq to other enzymes", in Kieleczawa J: DNA Sequencing II: Optimizing Preparation and Cleanup. Jones and Bartlett, pp. 241-257. ISBN 0-7637338-3-0. 
  13. ^ Myriam Vincent, Yan Xu and Huimin Kong (2004). "Helicase-dependent isothermal DNA amplification". EMBO reports 5 (8): 795–800. 
  14. ^ Q. Chou, M. Russell, D.E. Birch, J. Raymond and W. Bloch (1992). "Prevention of pre-PCR mis-priming and primer dimerization improves low-copy-number amplifications". Nucleic Acids Research 20: 1717-1723. 
  15. ^ E. Zietkiewicz, A. Rafalski, and D. Labuda (1994). "Genome fingerprinting by simple sequence repeat (SSR)-anchored polymerase chain reaction amplification". Genomics 20 (2): 176-83. 
  16. ^ Herman JG, Graff JR, Myöhänen S, Nelkin BD, Baylin SB (1996). "Methylation-specific PCR: a novel PCR assay for methylation status of CpG islands". Proc Natl Acad Sci U S A 93 (13): 9821-9826. PMID 8790415. 
  17. ^ Y.G. Liu and R. F. Whittier (1995). "Thermal asymmetric interlaced PCR: automatable amplification and sequencing of insert end fragments from P1 and YAC clones for chromosome walking". Genomics 25 (3): 674-81. 
  18. ^ David, F.Turlotte, E., (1998). "An Isothermal Amplification Method". C.R.Acad. Sci Paris, Life Science 321 (1): 909-914. 
  19. ^ http://www.lab-rech-associatives.com/pdf/Utiliser%20la%20Topologie%20de%20l'ADN.pdf

The Smithsonian Institution Building or Castle on the National Mall serves as the Institutions headquarters. ... Kary Banks Mullis, Ph. ... This article or section does not cite its references or sources. ...

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Image File history File links Commons-logo. ... Scientific American is a popular-science magazine, published (first weekly and later monthly) since August 28, 1845, making it the oldest continuously published magazine in the United States. ... The Cold Spring Harbor Laboratory (CSHL) is a research and educational institution, consisting of science laboratories located in Cold Spring Harbor, New York on Long Island, USA. The Laboratory has research programs focusing on cancer, neurobiology, plant genetics, genomics and bioinformatics, and has a broad educational mission, including the recently...


  Results from FactBites:
 
Polymerase Chain Reaction (PCR) (4059 words)
Polymerase chain reaction (PCR) is a molecular biology technique for enzymatically replicating DNA without using a living organism, such as E.
PCR is commonly used in medical and biological research labs for a variety of tasks, such as the detection of hereditary diseases, the identification of genetic fingerprints, the diagnosis of infectious diseases, the cloning of genes, paternity testing, and DNA computing.
PCR techniques have been successfully used on animals, such as a forty-thousand-year-old mammoth, and also on human DNA, in applications ranging from the analysis of Egyptian mummies to the identification of a Russian Tsar.
  More results at FactBites »

 
 

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