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A strip of eight PCR tubes, each containing a 100 μl reaction
.In molecular biology, the polymerase chain reaction (PCR) is a technique to amplify a single or few copies of a piece of DNA across several orders of magnitude, generating thousands to millions of copies of a particular DNA sequence.^ In the 1980s, he invented the polymerase chain reaction (PCR), a central technique in molecular biology which allows the amplification of specified DNA sequences.

^ Light-triggered polymerase chain reaction .
  • Light-triggered polymerase chain reaction (DOI: 10.1039/b715152g) 16 January 2010 13:52 UTC www.rsc.org [Source type: Academic]

^ PCR, polymerase chain reaction; Ig, immunoglobulin; NA, not available.
  • CDC - Real-Time Polymerase Chain Reaction for Detecting SARS Coronavirus, Beijing, 2003 16 January 2010 13:52 UTC www.cdc.gov [Source type: Academic]

.The method relies on thermal cycling, consisting of cycles of repeated heating and cooling of the reaction for DNA melting and enzymatic replication of the DNA. Primers (short DNA fragments) containing sequences complementary to the target region along with a DNA polymerase (after which the method is named) are key components to enable selective and repeated amplification.^ A target DNA fragment and its sequence-coincident DNA fragment interact with each other.
  • Multiplex polymerase chain reaction (PCR) with color-tagged module-shuffling primers for comparing gene expression levels in various cells -- Uematsu et al. 29 (16): e84 -- Nucleic Acids Research 16 January 2010 13:52 UTC nar.oxfordjournals.org [Source type: Academic]

^ The DNA fragment to be amplified is determined by selecting primers.
  • Polymerase chain reaction - Biocrawler, the free encyclopedia 17 January 2010 19:019 UTC www.biocrawler.com [Source type: Academic]

^ Presentation of the target DNA, primers and probes sequences.
  • PLoS Neglected Tropical Diseases: T. cruzi OligoC-TesT: A Simplified and Standardized Polymerase Chain Reaction Format for Diagnosis of Chagas Disease 16 January 2010 13:52 UTC www.plosntds.org [Source type: Academic]

.As PCR progresses, the DNA generated is itself used as a template for replication, setting in motion a chain reaction in which the DNA template is exponentially amplified.^ DNA itself is a chain of nucleotides.
  • The polymerase chain reaction -- Powledge 28 (2): 44 -- Advances in Physiology Education 16 January 2010 13:52 UTC advan.physiology.org [Source type: Academic]

^ In the polymerase chain reaction, a DNA template is repetitively: .
  • Bio572: The Polymerase Chain Reaction 16 January 2010 13:52 UTC www.escience.ws [Source type: FILTERED WITH BAYES]

^ This is because PCR amplification is a exponential reaction.
  • Real Time PCR Tutorial 17 January 2010 19:019 UTC pathmicro.med.sc.edu [Source type: FILTERED WITH BAYES]

.PCR can be extensively modified to perform a wide array of genetic manipulations.^ 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 .
  • Polymerase chain reaction - Hwiki 16 January 2010 13:52 UTC hwiki.fzk.de [Source type: Academic]

^ Since the PCR test for TPMT genotyping is not commercially established in Korea, we performed a sensitivity analysis with a wide range of values for PCR cost.
  • Pharmacoeconomic analysis of thiopurine methyltransferase polymorphism screening by polymerase chain reaction for treatment with azathioprine in Korea -- Oh et al. 43 (2): 156 -- Rheumatology 16 January 2010 13:52 UTC rheumatology.oxfordjournals.org [Source type: Academic]

^ All pre-PCR manipulations were performed in a laminar-flow cabinet using plugged pipette tips.
  • Quantitative Polymerase Chain Reaction for the Detection of Micrometastases in Patients With Breast Cancer -- Slade et al. 17 (3): 870 -- Journal of Clinical Oncology 16 January 2010 13:52 UTC jco.ascopubs.org [Source type: Academic]

.Almost all PCR applications employ a heat-stable DNA polymerase, such as Taq polymerase, an enzyme originally isolated from the bacterium Thermus aquaticus.^ A DNA polymerase is an enzyme that assists in DNA replication.

^ RNA can be amplified by Taq DNA polymerase.

^ One of the first thermostable DNA polymerases was obtained from Thermus aquaticus and was called "Taq."
  • Polymerase chain reaction - Hwiki 16 January 2010 13:52 UTC hwiki.fzk.de [Source type: Academic]

.This DNA polymerase enzymatically assembles a new DNA strand from DNA building blocks, the nucleotides, by using single-stranded DNA as a template and DNA oligonucleotides (also called DNA primers), which are required for initiation of DNA synthesis.^ For this reason DNA polymerase needs a primer at which it can add the first nucleotide.

^ DNA polymerase can only synthesize new DNA from the 5 to 3 (of the new DNA).

^ Primers are single-stranded.
  • The polymerase chain reaction -- Powledge 28 (2): 44 -- Advances in Physiology Education 16 January 2010 13:52 UTC advan.physiology.org [Source type: Academic]

.The vast majority of PCR methods use thermal cycling, i.e., alternately heating and cooling the PCR sample to a defined series of temperature steps.^ (If not using heated lid on PCR-block).
  • PCR trouble shooting, help, suggestions and advice 17 January 2010 19:019 UTC www.bio.uio.no [Source type: FILTERED WITH BAYES]

^ When used in conjunction with molecular beacons, LATE-PCR results in increased signal intensity and reduced sample variation.
  • Glossary of Real-Time PCR Terms [M.Tevfik DORAK] 17 January 2010 19:019 UTC dorakmt.tripod.com [Source type: Academic]

^ Therefore, samples that were PCR negative for viral genome but positive for K- ras were assumed to be without viral genome in the sample, or the viral load was assumed to be below the sensitivity of the method.
  • Tracheal Aspirate as a Substrate for Polymerase Chain Reaction Detection of Viral Genome in Childhood Pneumonia and Myocarditis -- Akhtar et al. 99 (15): 2011 -- Circulation 16 January 2010 13:52 UTC circ.ahajournals.org [Source type: Academic]

.These thermal cycling steps are necessary first to physically separate the two strands in a DNA double helix at a high temperature in a process called DNA melting.^ The two double-stranded DNA molecules (DNA ladders) separate into four strands.

^ The double-stranded DNA has to be heated to 94-96C in order to separate the strands.

^ Its function is to temporarily separate the two strands of a DNA double helix so that DNA or RNA synthesis can take place.

.At a lower temperature, each strand is then used as the template in DNA synthesis by the DNA polymerase to selectively amplify the target DNA. The selectivity of PCR results from the use of primers that are complementary to the DNA region targeted for amplification under specific thermal cycling conditions.^ Thus, the region of DNA to be synthesized is defined by the primers, which specifically anneal themselves to their complementary sequences on template strand, limiting the DNA fragment that will be amplified 9,46 .
  • Journal of Applied Oral Science - Reverse transcription and polymerase chain reaction: principles and applications in dentistry 16 January 2010 13:52 UTC www.scielo.br [Source type: Academic]

^ PCR is used to amplify a short, well-defined part of a DNA strand.

^ PCR is an in vitro method for amplifying DNA sequences using defined oligonucleotide primers Oligonucleotide primers A and B are complementary to DNA sequences located on opposite DNA strands and flanking the region to be amplified.
  • PCR, DNA sequencing and in vitro mutagenesis -- Human Molecular Genetics 2 -- NCBI Bookshelf 17 January 2010 19:019 UTC www.ncbi.nlm.nih.gov [Source type: Academic]

.Developed in 1983 by Kary Mullis,[1] PCR is now a common and often indispensable technique used in medical and biological research labs for a variety of applications.^ MeSH, 1991 Originally described in 1984 by Kary B. Mullis, who shared the Nobel Prize for Chemistry for this invention in 1993, PCR enables the amplification of specific nucleotide sequences through the use of a DNA polymerase.
  • PCR Polymerase Chain Reaction glossary & taxonomy 17 January 2010 19:019 UTC www.genomicglossaries.com [Source type: Academic]

^ ISBN: 9780849311840 Publication Date: November 13, 2003 Binding: Paperback A technique used to amplify the number of copies of a specific region of DNA, the polymerase chain reaction (PCR) is at the forefront of the dramatic development of biochemistry.
  • CRC Press Online - Book: The Polymerase Chain Reaction (PCR) for Human Viral Diagnosis 16 January 2010 13:52 UTC www.crcpress.com [Source type: Academic]

^ Kary Mullis, a molecular biologist working for Cetus Corporation, first conceived of the polymerase chain reaction (PCR) one evening in the spring of 1983 while driving his car down a California highway from San Francisco to Mendocino.
  • PCR AND CLONING: A TECHNOLOGY for the 21st CENTURY 17 January 2010 19:019 UTC www.sciencemag.org [Source type: Academic]

[2][3] .These include DNA cloning for sequencing, DNA-based phylogeny, or functional analysis of genes; the diagnosis of hereditary diseases; the identification of genetic fingerprints (used in forensic sciences and paternity testing); and the detection and diagnosis of infectious diseases.^ The use of gene cloning to amplify the cloned DNA and/or to produce lots of the cloned gene's product.
  • CHAPTER #10: GENETIC ENGINEERING 16 January 2010 13:52 UTC www.slic2.wsu.edu:82 [Source type: FILTERED WITH BAYES]

^ Detection of hereditary diseases .
  • Polymerase chain reaction - Hwiki 16 January 2010 13:52 UTC hwiki.fzk.de [Source type: Academic]

^ RT-PCR has found uses in gene expression studies, RNA sequence analysis, and diagnosis of infectious disease and genetic disorders.
  • PCR AND CLONING: A TECHNOLOGY for the 21st CENTURY 17 January 2010 19:019 UTC www.sciencemag.org [Source type: Academic]

In 1993, Mullis was awarded the Nobel Prize in Chemistry for his work on PCR.[4]

Contents

PCR principles and procedure

Figure 1a: An old thermal cycler for PCR
Figure 1b: An older model three-temperature thermal cycler for PCR
.PCR is used to amplify a specific region of a DNA strand (the DNA target).^ File:Pcr machine.jpg Figure 1 : A thermal cycler for PCR PCR is used to amplify specific regions of a DNA strand.
  • Polymerase chain reaction - Hwiki 16 January 2010 13:52 UTC hwiki.fzk.de [Source type: Academic]

^ PCR is used to amplify a short, well-defined part of a DNA strand.

^ When multiple primer sets were used, reproducible patterns of amplified complementary DNA fragments were obtained that showed strong dependence on sequence specificity of either primer.
  • Differential display of eukaryotic messenger RNA by means of the polymerase chain reaction -- Liang and Pardee 257 (5072): 967 -- Science 16 January 2010 13:52 UTC www.sciencemag.org [Source type: Academic]

.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.^ The result is a separation of the DNA fragments based on their length ( SIZE ).
  • CHAPTER #10: GENETIC ENGINEERING 16 January 2010 13:52 UTC www.slic2.wsu.edu:82 [Source type: FILTERED WITH BAYES]

^ PCR is an in vitro method for amplifying DNA (more...
  • PCR, DNA sequencing and in vitro mutagenesis -- Human Molecular Genetics 2 -- NCBI Bookshelf 17 January 2010 19:019 UTC www.ncbi.nlm.nih.gov [Source type: Academic]

^ Most PCR methods typically amplify DNA fragments of up to 10 kilo base pairs (kb).
  • Polymerase chain reaction - Hwiki 16 January 2010 13:52 UTC hwiki.fzk.de [Source type: Academic]

[5]
.A basic PCR set up requires several components and reagents.^ PCR, as currently practiced, requires several basic components {{ .
  • Polymerase chain reaction - Hwiki 16 January 2010 13:52 UTC hwiki.fzk.de [Source type: Academic]

^ The PCR reaction requires the following components: .
  • Polymerase Chain Reaction (PCR) 17 January 2010 19:019 UTC www.ncbi.nlm.nih.gov [Source type: Academic]

^ PCR, as currently practiced, requires several basic components.
  • What Is Pcr? 17 January 2010 19:019 UTC www.bionewsonline.com [Source type: Academic]
  • Polymerase chain reaction - Biocrawler, the free encyclopedia 17 January 2010 19:019 UTC www.biocrawler.com [Source type: Academic]

[6] These components include:
.The PCR is commonly carried out in a reaction volume of 10–200 μl in small reaction tubes (0.2–0.5 ml volumes) in a thermal cycler.^ 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 .
  • Polymerase chain reaction - Hwiki 16 January 2010 13:52 UTC hwiki.fzk.de [Source type: Academic]

^ Use 10 m l for 50 m l PCR reaction.
  • UNIT 8: POLYMERASE CHAIN REACTION (PCR) 16 January 2010 13:52 UTC www.med.unc.edu [Source type: Academic]

^ Typically, 10 to 200 μl of total reaction volume are used.
  • Polymerase chain reaction container and methods of using the same - Patent 5576197 16 January 2010 13:52 UTC www.freepatentsonline.com [Source type: Reference]

.The thermal cycler heats and cools the reaction tubes to achieve the temperatures required at each step of the reaction (see below).^ Run reaction in 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.

^ Researchers can program the heating element used for the annealing step to create a thermal gradient across the block, so the temperature of each column of reaction tubes varies by as much as 2° C. The RoboCycler ® Gradient 40 can maintain a gradient of up to 14° C; the maximum gradient for the RoboCycler ® Gradient 96 is 22° C. .
  • SCIENCE ELECTRONIC MARKETPLACE: STATISTICAL SOFTWARE SUPPLEMENT 17 January 2010 19:019 UTC www.sciencemag.org [Source type: FILTERED WITH BAYES]

.Many modern thermal cyclers make use of the Peltier effect which permits both heating and cooling of the block holding the PCR tubes simply by reversing the electric current.^ Peltier element : The element used for heating and cooling in a qPCR machine.
  • Glossary of Real-Time PCR Terms [M.Tevfik DORAK] 17 January 2010 19:019 UTC dorakmt.tripod.com [Source type: Academic]

^ The claimed container can be used for reverse transcription PCR in one tube.
  • Polymerase chain reaction container and methods of using the same - Patent 5576197 16 January 2010 13:52 UTC www.freepatentsonline.com [Source type: Reference]

^ (If not using heated lid on PCR-block).
  • PCR trouble shooting, help, suggestions and advice 17 January 2010 19:019 UTC www.bio.uio.no [Source type: FILTERED WITH BAYES]

.Thin-walled reaction tubes permit favorable thermal conductivity to allow for rapid thermal equilibration.^ Also, it is common to use "thin walled" tubes for polymerase chain reactions.
  • Bio572: The Polymerase Chain Reaction 16 January 2010 13:52 UTC www.escience.ws [Source type: FILTERED WITH BAYES]

^ PCR tubes thin wall, w/flat..
  • Gene Expression : PCR : bioWORLD - Serving Life-Sciences & Biotechnology Industries Worldwide 17 January 2010 19:019 UTC www.bio-world.com [Source type: FILTERED WITH BAYES]

^ Thin-walled reaction tubes permit favorable thermal conductivity to allow for rapid thermal equilibration.
  • PCR | Marlow Industries 17 January 2010 19:019 UTC www.marlow.com [Source type: Academic]
  • Polymerase Chain Reaction (PCR) K-12 Experiments and Background Information 16 January 2010 13:52 UTC www.juliantrubin.com [Source type: Academic]

.Most thermal cyclers have heated lids to prevent condensation at the top of the reaction tube.^ Most thermal cyclers have heated lids to prevent condensation at the top of the reaction tube.
  • Polymerase Chain Reaction (PCR) K-12 Experiments and Background Information 16 January 2010 13:52 UTC www.juliantrubin.com [Source type: Academic]

^ Do not touch the top of reaction mixture in the tubes.
  • 20-mer Polymerase Chain Reaction Procedure (for MJ Research Thermal Cycler) 16 January 2010 13:52 UTC wheat.pw.usda.gov [Source type: Academic]

^ This step may be omitted if the thermal cycler is equipped with a heated lid.
  • PCR - IGEM07 17 January 2010 19:019 UTC parts.mit.edu [Source type: Academic]

.Older thermocyclers lacking a heated lid require a layer of oil on top of the reaction mixture or a ball of wax inside the tube.^ Older thermocyclers lacking a heated lid require a layer of oil on top of the reaction mixture or a ball of wax inside the tube.
  • Polymerase Chain Reaction (PCR) K-12 Experiments and Background Information 16 January 2010 13:52 UTC www.juliantrubin.com [Source type: Academic]

^ Most thermal cyclers have heated lids to prevent condensation at the top of the reaction tube.
  • Polymerase Chain Reaction (PCR) K-12 Experiments and Background Information 16 January 2010 13:52 UTC www.juliantrubin.com [Source type: Academic]

^ Add 100ul of mineral oil on top of the reaction mixtures and place the tubes on ice.
  • AMBL » Amplification of a specific DNA sequence by Polymerase Chain Reaction (PCR) 16 January 2010 13:52 UTC www.bioteach.ubc.ca [Source type: Academic]

Procedure

.
Figure 2: Schematic drawing of the PCR cycle.
^ File:Pcr.png Figure 2 : Schematic drawing of the PCR cycle.
  • Polymerase chain reaction - Hwiki 16 January 2010 13:52 UTC hwiki.fzk.de [Source type: Academic]

^ The figure shows that the MSP-PCR method has a useful dynamic range from 0.1 to 10.0 amol at 30 thermal cycles.
  • Multiplex polymerase chain reaction (PCR) with color-tagged module-shuffling primers for comparing gene expression levels in various cells -- Uematsu et al. 29 (16): e84 -- Nucleic Acids Research 16 January 2010 13:52 UTC nar.oxfordjournals.org [Source type: Academic]

^ The direct quantitative RT-PCR method is outlined schematically in Figure 2 .
  • BioMed Central | Full text | A new reverse transcription-polymerase chain reaction method for accurate quantification 16 January 2010 13:52 UTC www.biomedcentral.com [Source type: Academic]

.(1) Denaturing at 94–96 °C. (2) Annealing at ~65 °C (3) Elongation at 72 °C.^ In both reactions, the PCR conditions were 22 cycles of denaturation (98 C, 10 sec), annealing (65 C, 30 sec), and extension (72 C, 90 sec; autosegment extension, 5 sec).
  • Detection of Monoclonality of the Immunoglobulin Heavy Chain Gene in Thyroid Malignant Lymphoma by Vectorette Polymerase Chain Reaction -- Takano et al. 90 (2): 720 -- Journal of Clinical Endocrinology & Metabolism 16 January 2010 13:52 UTC jcem.endojournals.org [Source type: Academic]

^ The first denaturing cycle was at 95°C for 3 minutes, followed by 30 cycles of denaturation at 94°C for 1 minute, annealing at 60°C for 1 minute, and extension at 72°C for 2 minutes.
  • Polymerase Chain Reaction-Based Method for Quantifying Recruitment of Monocytes to Mouse Atherosclerotic Lesions In Vivo : Enhancement by Tumor Necrosis Factor-{alpha} and Interleukin-1{beta} -- Kim et al. 20 (8): 1976 -- Arteriosclerosis, Thrombosis, and Vascular Biology 16 January 2010 13:52 UTC atvb.ahajournals.org [Source type: Academic]

^ Denaturing at 94-96°C. (2) Annealing at (eg) 68°C. (3) Elongation at 72°C (P=Polymerase).
  • Polymerase chain reaction - Hwiki 16 January 2010 13:52 UTC hwiki.fzk.de [Source type: Academic]

Four cycles are shown here. The blue lines represent the DNA template to which primers (red arrows) anneal that are extended by the DNA polymerase (light green circles), to give shorter DNA products (green lines), which themselves are used as templates as PCR progresses.
.The PCR usually consists of a series of 20-40 repeated temperature changes called cycles; each cycle typically consists of 2-3 discrete temperature steps.^ The PCR usually consists of a series of 20 to 35 cycles.
  • Polymerase chain reaction - Hwiki 16 January 2010 13:52 UTC hwiki.fzk.de [Source type: Academic]

^ This cycle is usually repeated 30 times.

^ The PCR process consists of the following steps: .
  • Polymerase chain reaction - Hwiki 16 January 2010 13:52 UTC hwiki.fzk.de [Source type: Academic]

.Most commonly PCR is carried out with cycles that have three temperature steps (Fig.^ Most commonly PCR is carried out with cycles that have three temperature steps.
  • Polymerase Chain Reaction (PCR) K-12 Experiments and Background Information 16 January 2010 13:52 UTC www.juliantrubin.com [Source type: Academic]

^ Hybridization is carried out as illustrated in Fig.
  • CHAPTER #10: GENETIC ENGINEERING 16 January 2010 13:52 UTC www.slic2.wsu.edu:82 [Source type: FILTERED WITH BAYES]

^ There are three basic steps in PCR: .
  • PCR - The science behind DNA testing 17 January 2010 19:019 UTC www.dnajunction.com [Source type: Academic]

2). .The cycling is often preceded by a single temperature step (called hold) at a high temperature (>90°C), and followed by one hold at the end for final product extension or brief storage.^ The cycling is often preceded by a single temperature step (called hold) at a high temperature (> 90°C), and followed by one hold at the end for final product extension or brief storage.
  • Polymerase Chain Reaction (PCR) K-12 Experiments and Background Information 16 January 2010 13:52 UTC www.juliantrubin.com [Source type: Academic]

^ In the first step, the denaturation, the temperature is raised to above 90° C. Due to the high temperature, the hydrogen bonds between the two strands of DNA break and the strands are separated.
  • Polymerase chain reaction - encyclopedia article - Citizendium 16 January 2010 13:52 UTC en.citizendium.org [Source type: Academic]

^ During PCR, the template DNA is denatured to single-strands at a high temperature, then allowed to anneal with an excess of primers at a lower temperature, followed by synthesis of DNA or RNA from the primers at a temperature optimum for the specific polymerase enzyme.
  • AMBL » Amplification of a specific DNA sequence by Polymerase Chain Reaction (PCR) 16 January 2010 13:52 UTC www.bioteach.ubc.ca [Source type: Academic]

.The temperatures used and the length of time they are applied in each cycle depend on a variety of parameters.^ Cycle times and temperatures .

^ The temperatures used and the length of time they are applied in each cycle depend on a variety of parameters.
  • Polymerase Chain Reaction (PCR) K-12 Experiments and Background Information 16 January 2010 13:52 UTC www.juliantrubin.com [Source type: Academic]

^ Elongation or extension: Phase of PCR cycle following annealing of primer during which the Taq polymerase synthesizes a strand of DNA. The optimum temperature depends on the enzyme used but is usually between 68-72?
  • MMIA 17 January 2010 19:019 UTC www.iupui.edu [Source type: Academic]

.These include the enzyme used for DNA synthesis, the concentration of divalent ions and dNTPs in the reaction, and the melting temperature (Tm) of the primers.^ The G+C content of the primer will affect the reaction temperature.
  • UNIT 8: POLYMERASE CHAIN REACTION (PCR) 16 January 2010 13:52 UTC www.med.unc.edu [Source type: Academic]

^ The melting temperature increases with the length of the primer.

^ When multiple primer sets were used, reproducible patterns of amplified complementary DNA fragments were obtained that showed strong dependence on sequence specificity of either primer.
  • Differential display of eukaryotic messenger RNA by means of the polymerase chain reaction -- Liang and Pardee 257 (5072): 967 -- Science 16 January 2010 13:52 UTC www.sciencemag.org [Source type: Academic]

[8]
.
  • Initialization step: This step consists of heating the reaction to a temperature of 94–96 °C (or 98 °C if extremely thermostable polymerases are used), which is held for 1–9 minutes.^ Reaction mixtures may be heated at 100° C. for two minutes prior to addition of Taq DNA polymerase (2.5 U, Perkin-Elmer Cetus, Connecticut), overlaid with 100 μl of light mineral oil, and subjected to 30 cycles consisting of a denaturation step (1 minute, 94° C.), primer annealing (30 seconds, 45° C.) and DNA synthesis (30 seconds, 72° C.) using, for example, a Perkin Elmer Cetus DNA thermal cycler.
    • Polymerase chain reaction assays for monitoring antiviral therapy and making therapeutic decisions in the treatment of acquired immunodeficiency syndrome - Patent 5650268 16 January 2010 13:52 UTC www.freepatentsonline.com [Source type: Academic]

    ^ An initial denaturation step at 94°C for 15 minutes to activate the Hot Star Taq polymerase was followed by 40 cycles of 94°C for 20 seconds, 65°C for 20 seconds and 72°C for 20 seconds, and a final extension at 72°C for 1 minute.
    • PLoS Neglected Tropical Diseases: T. cruzi OligoC-TesT: A Simplified and Standardized Polymerase Chain Reaction Format for Diagnosis of Chagas Disease 16 January 2010 13:52 UTC www.plosntds.org [Source type: Academic]

    ^ Those techniques present a problem, says Janoson, because two-step methods require different buffers and temperatures to perform RT-PCR. The researcher first performs the reverse transcriptase step in one tube; stops the reaction; then adds different components, including a thermostable DNA polymerase, for the PCR step.
    • SCIENCE ELECTRONIC MARKETPLACE: STATISTICAL SOFTWARE SUPPLEMENT 17 January 2010 19:019 UTC www.sciencemag.org [Source type: FILTERED WITH BAYES]

    .It is only required for DNA polymerases that require heat activation by hot-start PCR.^ "Hot starts" refer to the addition of the polymerase after the DNA has denatured.
    • PCR 17 January 2010 19:019 UTC www.omrf.org [Source type: Academic]

    ^ Hot-start PCR .
    • PCR, DNA sequencing and in vitro mutagenesis -- Human Molecular Genetics 2 -- NCBI Bookshelf 17 January 2010 19:019 UTC www.ncbi.nlm.nih.gov [Source type: Academic]

    ^ DNA polymerase can only synthesize new DNA from the 5 to 3 (of the new DNA).

    [9]
  • .
  • Denaturation step: This step is the first regular cycling event and consists of heating the reaction to 94–98 °C for 20–30 seconds.^ In the first step of a PCR reaction the DNA is denatured by heating to 93 o C to 94 o C so that the strands come apart.
    • UNIT 8: POLYMERASE CHAIN REACTION (PCR) 16 January 2010 13:52 UTC www.med.unc.edu [Source type: Academic]

    ^ Reaction mixtures may be heated at 100° C. for two minutes prior to addition of Taq DNA polymerase (2.5 U, Perkin-Elmer Cetus, Connecticut), overlaid with 100 μl of light mineral oil, and subjected to 30 cycles consisting of a denaturation step (1 minute, 94° C.), primer annealing (30 seconds, 45° C.) and DNA synthesis (30 seconds, 72° C.) using, for example, a Perkin Elmer Cetus DNA thermal cycler.
    • Polymerase chain reaction assays for monitoring antiviral therapy and making therapeutic decisions in the treatment of acquired immunodeficiency syndrome - Patent 5650268 16 January 2010 13:52 UTC www.freepatentsonline.com [Source type: Academic]

    ^ An initial denaturation step at 94°C for 15 minutes to activate the Hot Star Taq polymerase was followed by 40 cycles of 94°C for 20 seconds, 65°C for 20 seconds and 72°C for 20 seconds, and a final extension at 72°C for 1 minute.
    • PLoS Neglected Tropical Diseases: T. cruzi OligoC-TesT: A Simplified and Standardized Polymerase Chain Reaction Format for Diagnosis of Chagas Disease 16 January 2010 13:52 UTC www.plosntds.org [Source type: Academic]

    .It causes DNA melting of the DNA template by disrupting the hydrogen bonds between complementary bases, yielding single strands of DNA.
  • Annealing step: The reaction temperature is lowered to 50–65 °C for 20–40 seconds allowing annealing of the primers to the single-stranded DNA template.^ This removes the mRNA allowing the second strand of DNA to be formed.
    • Real Time PCR Tutorial 17 January 2010 19:019 UTC pathmicro.med.sc.edu [Source type: FILTERED WITH BAYES]

    ^ The second temperature, typically about low 60° C., anneals the primer to the single strand target.
    • Polymerase chain reaction container and methods of using the same - Patent 5576197 16 January 2010 13:52 UTC www.freepatentsonline.com [Source type: Reference]

    ^ Annealed primers are incorporated into the newly synthesized DNA strands.
    • PCR, DNA sequencing and in vitro mutagenesis -- Human Molecular Genetics 2 -- NCBI Bookshelf 17 January 2010 19:019 UTC www.ncbi.nlm.nih.gov [Source type: Academic]

    .Typically the annealing temperature is about 3-5 degrees Celsius below the Tm of the primers used.^ A typical annealing temperature one might use is 50 to 60 degrees Celsius.
    • Bio572: The Polymerase Chain Reaction 16 January 2010 13:52 UTC www.escience.ws [Source type: FILTERED WITH BAYES]

    ^ For every subsequent cycle, the annealing temperature is decreased by 1 degree Celsius.

    ^ This is particularly useful when testing suitable annealing temperatures for primers.

    .Stable DNA-DNA hydrogen bonds are only formed when the primer sequence very closely matches the template sequence.^ In DNA, these bases form hydrogen bonds with their complementary purines.

    ^ The stability of the primer-template DNA duplex can be measured by the melting temperature (T m ).
    • Primer Design Guide for PCR :: Learn Designing Primers for PCR 17 January 2010 19:019 UTC www.premierbiosoft.com [Source type: Academic]

    ^ Primer presentation on the DNA sequence: .
    • Online Analysis Tools - PCR 17 January 2010 19:019 UTC molbiol-tools.ca [Source type: Academic]

    .The polymerase binds to the primer-template hybrid and begins DNA synthesis.
  • Extension/elongation step: The temperature at this step depends on the DNA polymerase used; Taq polymerase has its optimum activity temperature at 75–80 °C,[10][11] and commonly a temperature of 72 °C is used with this enzyme.^ The process is based on a temperature-dependent reversible inhibitor of Taq polymerase.
    • Brinkmann Instruments Canada ::: FAQs-PCR 17 January 2010 19:019 UTC www.brinkmanncanada.com [Source type: FILTERED WITH BAYES]

    ^ A DNA polymerase is an enzyme that assists in DNA replication.

    ^ Raising the temperature again (typically to 72C) stimulates the heat-stable Taq polymerase to copy the DNA template in a further extension step.
    • Journal of Applied Oral Science - Reverse transcription and polymerase chain reaction: principles and applications in dentistry 16 January 2010 13:52 UTC www.scielo.br [Source type: Academic]

    .At this step the DNA polymerase synthesizes a new DNA strand complementary to the DNA template strand by adding dNTPs that are complementary to the template in 5' to 3' direction, condensing the 5'-phosphate group of the dNTPs with the 3'-hydroxyl group at the end of the nascent (extending) DNA strand.^ Now DNA polymerase can recognize the start of the gene and rebuild the complementary strand in the 5' to 3' direction.

    ^ DNA polymerase can only synthesize new DNA from the 5 to 3 (of the new DNA).

    ^ 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.
    • Polymerase chain reaction - Hwiki 16 January 2010 13:52 UTC hwiki.fzk.de [Source type: Academic]

    .The extension time depends both on the DNA polymerase used and on the length of the DNA fragment to be amplified.^ Thermostable DNA polymerase with enhanced thermostability and enhanced length and efficiency of primer extension.

    ^ RNA can be amplified by Taq DNA polymerase.

    ^ The time for this step depends both on the DNA-Polymerase itself and on the length of the DNA fragment to be amplified.
    • What Is Pcr? 17 January 2010 19:019 UTC www.bionewsonline.com [Source type: Academic]
    • Polymerase chain reaction - Biocrawler, the free encyclopedia 17 January 2010 19:019 UTC www.biocrawler.com [Source type: Academic]

    .As a rule-of-thumb, at its optimum temperature, the DNA polymerase will polymerize a thousand bases per minute.^ As a rule-of-thumb we use 1 minute per 1000bp.

    ^ The elongation temperature depends on the DNA-Polymerase.

    ^ DNA SOLUBILIZATION Remove the ethanol wash and briefly air-dry the DNA pellet by keeping tubes open for 3 - 5 minutes at room temperature.
    • Molecular Research Center RNA & DNA isolation reagents for RT-PCR, PCR, microarrays 17 January 2010 19:019 UTC www.mrcgene.com [Source type: Academic]

    Under optimum conditions, i.e., if there are no limitations due to limiting substrates or reagents, at each extension step, the amount of DNA target is doubled, leading to exponential (geometric) amplification of the specific DNA fragment.
  • Final elongation: This single step is occasionally performed at a temperature of 70–74 °C for 5–15 minutes after the last PCR cycle to ensure that any remaining single-stranded DNA is fully extended.
  • Final hold: This step at 4–15 °C for an indefinite time may be employed for short-term storage of the reaction.
.
Figure 3: Ethidium bromide-stained PCR products after gel electrophoresis.
^ After amplification, the products are size-fractionated by electrophoresis on an agarose or polyacrylamide gel stained with ethidium bromide.
  • Journal of Applied Oral Science - Reverse transcription and polymerase chain reaction: principles and applications in dentistry 16 January 2010 13:52 UTC www.scielo.br [Source type: Academic]

^ The gel is stained with ethidium bromide .
  • Real Time PCR Tutorial 17 January 2010 19:019 UTC pathmicro.med.sc.edu [Source type: FILTERED WITH BAYES]

^ The PCR products can then be size-fractionated by polyacrylamide gel electrophoresis.
  • PCR, DNA sequencing and in vitro mutagenesis -- Human Molecular Genetics 2 -- NCBI Bookshelf 17 January 2010 19:019 UTC www.ncbi.nlm.nih.gov [Source type: Academic]

.Two sets of primers were used to amplify a target sequence from three different tissue samples.^ Nested primers are complementary to sequences in the target that are different from the primers in the initial amplification.
  • Polymerase chain reaction container and methods of using the same - Patent 5576197 16 January 2010 13:52 UTC www.freepatentsonline.com [Source type: Reference]

^ Two primers: to flank the target sequence.
  • PCR (Polymerase Chain Reaction) 16 January 2010 13:52 UTC www.slideshare.net [Source type: Reference]

^ Primers for this normalization standard do not amplify sequences from any of the environmental sample tested.
  • Analysis of Specific Bacteria from Environmental Samples using a Quantitative Polymerase Chain Reaction 16 January 2010 13:52 UTC www.open-access-biology.com [Source type: Academic]

.No amplification is present in sample #1; DNA bands in sample #2 and #3 indicate successful amplification of the target sequence.^ Presentation of the target DNA, primers and probes sequences.
  • PLoS Neglected Tropical Diseases: T. cruzi OligoC-TesT: A Simplified and Standardized Polymerase Chain Reaction Format for Diagnosis of Chagas Disease 16 January 2010 13:52 UTC www.plosntds.org [Source type: Academic]

^ A target DNA sequence that is not present in the environmental samples is the internal normalization standard.
  • Analysis of Specific Bacteria from Environmental Samples using a Quantitative Polymerase Chain Reaction 16 January 2010 13:52 UTC www.open-access-biology.com [Source type: Academic]

^ A sample of the TARGET DNA to be copied.
  • CHAPTER #10: GENETIC ENGINEERING 16 January 2010 13:52 UTC www.slic2.wsu.edu:82 [Source type: FILTERED WITH BAYES]

The gel also shows a positive control, and a DNA ladder containing DNA fragments of defined length for sizing the bands in the experimental PCRs.
.To check whether the PCR generated the anticipated DNA fragment (also sometimes referred to as the amplimer or amplicon), agarose gel electrophoresis is employed for size separation of the PCR products.^ The PCR products can then be size-fractionated by polyacrylamide gel electrophoresis.
  • PCR, DNA sequencing and in vitro mutagenesis -- Human Molecular Genetics 2 -- NCBI Bookshelf 17 January 2010 19:019 UTC www.ncbi.nlm.nih.gov [Source type: Academic]

^ The product was analyzed on a 1% agarose gel.
  • PLoS ONE: Evolution in Quantum Leaps: Multiple Combinatorial Transfers of HPI and Other Genetic Modules in Enterobacteriaceae 17 January 2010 19:019 UTC www.plosone.org [Source type: Academic]

^ The cut-DNA is separated into fragments according to SIZE on a gel.
  • CHAPTER #10: GENETIC ENGINEERING 16 January 2010 13:52 UTC www.slic2.wsu.edu:82 [Source type: FILTERED WITH BAYES]

.The size(s) of PCR products is determined by comparison with a DNA ladder (a molecular weight marker), which contains DNA fragments of known size, run on the gel alongside the PCR products (see Fig.^ The PCR products can then be size-fractionated by polyacrylamide gel electrophoresis.
  • PCR, DNA sequencing and in vitro mutagenesis -- Human Molecular Genetics 2 -- NCBI Bookshelf 17 January 2010 19:019 UTC www.ncbi.nlm.nih.gov [Source type: Academic]

^ The cut-DNA is separated into fragments according to SIZE on a gel.
  • CHAPTER #10: GENETIC ENGINEERING 16 January 2010 13:52 UTC www.slic2.wsu.edu:82 [Source type: FILTERED WITH BAYES]

^ The size of the PCR product can be determined by comparing it with a DNA ladder , which contains DNA fragments of known size, also loaded onto the gel (Fig.
  • Polymerase chain reaction - Hwiki 16 January 2010 13:52 UTC hwiki.fzk.de [Source type: Academic]

3).

PCR stages

The PCR process can be divided into three stages:
.Exponential amplification: At every cycle, the amount of product is doubled (assuming 100% reaction efficiency).^ This is because PCR amplification is a exponential reaction.
  • Real Time PCR Tutorial 17 January 2010 19:019 UTC pathmicro.med.sc.edu [Source type: FILTERED WITH BAYES]

^ LATE-PCR begins with an exponential phase in which amplification efficiency is similar to that of symmetric PCR. Once the limiting primer is depleted, the reaction abruptly switches to linear amplification, and the single-stranded product is made for many additional thermal cycles.
  • Glossary of Real-Time PCR Terms [M.Tevfik DORAK] 17 January 2010 19:019 UTC dorakmt.tripod.com [Source type: Academic]

^ Earlier on we derived the equation that the change in amount of DNA after n cycles is equal to the efficiency to the power of n.
  • Real Time PCR Tutorial 17 January 2010 19:019 UTC pathmicro.med.sc.edu [Source type: FILTERED WITH BAYES]

.The reaction is very sensitive: only minute quantities of DNA need to be present.^ For example, serologic and neurologic analyses for Lyme disease or multiple sclerosis need only be conducted if the patient presents with appropriate symptoms."
  • Polymerase Chain Reaction Testing: Selected Indications 16 January 2010 13:52 UTC www.aetna.com [Source type: Academic]

^ PCR optimisation Since PCR is very sensitive, adequate measures to avoid contamination from other DNA present in lab environment (bacteria, viruses, own DNA etc.

^ American Lyme Disease Foundation states: "The polymerase chain reaction (PCR) test is a very sensitive assay that detects the DNA of B. burgdorferi .
  • Polymerase Chain Reaction Testing: Selected Indications 16 January 2010 13:52 UTC www.aetna.com [Source type: Academic]

[12]
.Levelling off stage: The reaction slows as the DNA polymerase loses activity and as consumption of reagents such as dNTPs and primers causes them to become limiting.^ But with the new Taq Bead TM , the researcher simply puts one bead into each 0.5 ml reaction tube and adds the other reagents: the DNA template to be amplified; the primers; buffer; magnesium chloride; and the four deoxynucleotide triphosphates (dNTPs), adenine (A), thymine (T), guanine (G), and cytosine (C).
  • SCIENCE ELECTRONIC MARKETPLACE: STATISTICAL SOFTWARE SUPPLEMENT 17 January 2010 19:019 UTC www.sciencemag.org [Source type: FILTERED WITH BAYES]

^ In research studies, culture of saliva or peripheral blood mononuclear cells for EBV, in situ DNA hybridization, or polymerase chain reaction can determine the presence of EBV or EBV DNA and may implicate EBV with a syndrome, such as lymphoproliferation (AAP, 2006).
  • Polymerase Chain Reaction Testing: Selected Indications 16 January 2010 13:52 UTC www.aetna.com [Source type: Academic]

^ 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.

.Plateau: No more product accumulates due to exhaustion of reagents and enzyme.^ It requires no more than a test tube, a few simple reagents, and a source of heat."
  • Polymerase chain reaction - Hwiki 16 January 2010 13:52 UTC hwiki.fzk.de [Source type: Academic]

^ No amplification controls (NAC, a minus enzyme control): In mRNA analysis, NAC is a mock reverse transcription containing all the RT-PCR reagents, except the reverse transcriptase.
  • Glossary of Real-Time PCR Terms [M.Tevfik DORAK] 17 January 2010 19:019 UTC dorakmt.tripod.com [Source type: Academic]

PCR optimization

.In practice, PCR can fail for various reasons, in part due to its sensitivity to contamination causing amplification of spurious DNA products.^ Any amplification suggests genomic DNA contamination.
  • Glossary of Real-Time PCR Terms [M.Tevfik DORAK] 17 January 2010 19:019 UTC dorakmt.tripod.com [Source type: Academic]

^ Preparation of DNA, PCR amplification, and product analysis.
  • STORAGE DURATION AND POLYMERASE CHAIN REACTION DETECTION OF PLASMODIUM FALCIPARUM FROM BLOOD SPOTS ON FILTER PAPER -- CHAORATTANAKAWEE et al. 69 (1): 42 -- American Journal of Tropical Medicine and Hygiene 16 January 2010 13:52 UTC www.ajtmh.org [Source type: Academic]

^ The inherent sensitivity of PCR amplification can lead to amplification of nonspecific sequences and contaminants.
  • Utility of polymerase chain reaction as a diagnostic tool in cutaneous tuberculosis Padmavathy L, Rao L, Veliath A - Indian J Dermatol Venereol Leprol 16 January 2010 13:52 UTC www.ijdvl.com [Source type: Academic]

.Because of this, a number of techniques and procedures have been developed for optimizing PCR conditions.^ Recent developments in PCR techniques .
  • Polymerase chain reaction - Hwiki 16 January 2010 13:52 UTC hwiki.fzk.de [Source type: Academic]

^ Development and optimization of FQ-PCR .
  • Virology Journal | Full text | Development of a fluorescent quantitative real-time polymerase chain reaction assay for the detection of Goose parvovirus in vivo 16 January 2010 13:52 UTC www.virologyj.com [Source type: Academic]

^ Development and optimization of FQ-PCR and conventional PCR .
  • Virology Journal | Full text | Development of a fluorescent quantitative real-time polymerase chain reaction assay for the detection of Goose parvovirus in vivo 16 January 2010 13:52 UTC www.virologyj.com [Source type: Academic]

[13][14] .Contamination with extraneous DNA is addressed with lab protocols and procedures that separate pre-PCR mixtures from potential DNA contaminants.^ Any procedure in which a technician handles the PCR container has the potential to contaminate the sample.
  • Polymerase chain reaction container and methods of using the same - Patent 5576197 16 January 2010 13:52 UTC www.freepatentsonline.com [Source type: Reference]

^ PCR optimisation Since PCR is very sensitive, adequate measures to avoid contamination from other DNA present in lab environment (bacteria, viruses, own DNA etc.

^ PCR amplification and DNA sequencing of HIV-1 env sequences from the mother’s two and the child’s three culture supernatants were performed in separate laboratories to eliminate the possibility of cross-contamination.
  • Concerns about HIV/AIDS Testing and Measurement 16 January 2010 13:52 UTC rethinkaids.com [Source type: Academic]

[6] .This usually involves spatial separation of PCR-setup areas from areas for analysis or purification of PCR products, use of disposable plasticware, and thoroughly cleaning the work surface between reaction setups.^ Use 10 m l for 50 m l PCR reaction.
  • UNIT 8: POLYMERASE CHAIN REACTION (PCR) 16 January 2010 13:52 UTC www.med.unc.edu [Source type: Academic]

^ Two sequential analyses and manual processing are required, however, exposing the concentrated PCR product to the laboratory and increasing the chance of PCR product contamination of subsequent reactions.
  • ScienceDirect - Analytical Biochemistry : Quantitative heteroduplex analysis for single nucleotide polymorphism genotyping 17 January 2010 19:019 UTC www.math.utah.edu [Source type: Academic]

^ The pCR ® -Blunt vector works by positive selection; it allows only the growth of recombinants that contain the PCR product.
  • SCIENCE ELECTRONIC MARKETPLACE: STATISTICAL SOFTWARE SUPPLEMENT 17 January 2010 19:019 UTC www.sciencemag.org [Source type: FILTERED WITH BAYES]

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.

Application of PCR

Selective DNA isolation

.PCR allows isolation of DNA fragments from genomic DNA by selective amplification of a specific region of DNA. This use of PCR augments many methods, such as generating hybridization probes for Southern or northern hybridization and DNA cloning, which require larger amounts of DNA, representing a specific DNA region.^ PCR amplification was performed on isolates with Eh GI-1 used in this study.
  • PLoS ONE: Evolution in Quantum Leaps: Multiple Combinatorial Transfers of HPI and Other Genetic Modules in Enterobacteriaceae 17 January 2010 19:019 UTC www.plosone.org [Source type: Academic]

^ Any amplification suggests genomic DNA contamination.
  • Glossary of Real-Time PCR Terms [M.Tevfik DORAK] 17 January 2010 19:019 UTC dorakmt.tripod.com [Source type: Academic]

^ PCR is the enzymatic amplification of a specific DNA sequence in vitro 9 .
  • Journal of Applied Oral Science - Reverse transcription and polymerase chain reaction: principles and applications in dentistry 16 January 2010 13:52 UTC www.scielo.br [Source type: Academic]

.PCR supplies these techniques with high amounts of pure DNA, enabling analysis of DNA samples even from very small amounts of starting material.^ Template DNA or sample starting material .

^ Application of PCR: PCR allows isolation of DNA fragments from genomic DNA by selective amplification of a specific region, thus PCR supplies techniques with high amounts of pure DNA, enabling analysis of DNA samples even from very small amounts of starting material.
  • Polymerase Chain Reaction (PCR) K-12 Experiments and Background Information 16 January 2010 13:52 UTC www.juliantrubin.com [Source type: Academic]

^ Major role in the human genome project , replacing a single polymerase with a blend of a thermostable polymerase and a proofreading (Pwo DNA polymerase) made PCR an indispensable tool in the analysis and mapping of entire genomes by greatly extending the length of the sequence that could be amplified, increasing the amount of PCR product and providing higher fidelity during PCR. Identify the level of expression of genes in extremely small samples of material, e.g.
  • PCR (Polymerase Chain Reaction) 16 January 2010 13:52 UTC www.slideshare.net [Source type: Reference]

.Other applications of PCR include DNA sequencing to determine unknown PCR-amplified sequences in which one of the amplification primers may be used in Sanger sequencing, isolation of a DNA sequence to expedite recombinant DNA technologies involving the insertion of a DNA sequence into a plasmid or the genetic material of another organism.^ PCR amplification was performed on isolates with Eh GI-1 used in this study.
  • PLoS ONE: Evolution in Quantum Leaps: Multiple Combinatorial Transfers of HPI and Other Genetic Modules in Enterobacteriaceae 17 January 2010 19:019 UTC www.plosone.org [Source type: Academic]

^ A universal sequencing primer can be used to sequence (more...
  • PCR, DNA sequencing and in vitro mutagenesis -- Human Molecular Genetics 2 -- NCBI Bookshelf 17 January 2010 19:019 UTC www.ncbi.nlm.nih.gov [Source type: Academic]

^ PCR is the enzymatic amplification of a specific DNA sequence in vitro 9 .
  • Journal of Applied Oral Science - Reverse transcription and polymerase chain reaction: principles and applications in dentistry 16 January 2010 13:52 UTC www.scielo.br [Source type: Academic]

.Bacterial colonies (E.coli) can be rapidly screened by PCR for correct DNA vector constructs.^ An internal standard was constructed by the insertion of a 222-bp length of foreign DNA into the Xba I restriction site of the segment amplified by PCR. This fragment was ligated into pGEM-T vector (Promega).
  • Polymerase Chain Reaction-Based Method for Quantifying Recruitment of Monocytes to Mouse Atherosclerotic Lesions In Vivo : Enhancement by Tumor Necrosis Factor-{alpha} and Interleukin-1{beta} -- Kim et al. 20 (8): 1976 -- Arteriosclerosis, Thrombosis, and Vascular Biology 16 January 2010 13:52 UTC atvb.ahajournals.org [Source type: Academic]

^ So the researcher creates the mutant DNA, uses PCR to amplify it, and then screens the new sequence for an altered ability to bind to the protein.
  • SCIENCE ELECTRONIC MARKETPLACE: STATISTICAL SOFTWARE SUPPLEMENT 17 January 2010 19:019 UTC www.sciencemag.org [Source type: FILTERED WITH BAYES]

^ Because of its rapidity and simplicity, PCR is ideally suited to providing numerous DNA templates for mutation screening.
  • PCR, DNA sequencing and in vitro mutagenesis -- Human Molecular Genetics 2 -- NCBI Bookshelf 17 January 2010 19:019 UTC www.ncbi.nlm.nih.gov [Source type: Academic]

[15] .PCR may also be used for genetic fingerprinting; a forensic technique used to identify a person or organism by comparing experimental DNAs through different PCR-based methods.^ The PCR results were compared with the conventional culture based method of diagnosis.
  • Polymerase chain reaction based diagnosis of systemic fungal infections and sensitivity testing of the fungal isolates RS Iyer, RB Pal, RY Patel, DD Banker - Indian J Med Microbiol 16 January 2010 13:52 UTC www.ijmm.org [Source type: Academic]

^ Standard PCR-based DNA labeling .
  • PCR, DNA sequencing and in vitro mutagenesis -- Human Molecular Genetics 2 -- NCBI Bookshelf 17 January 2010 19:019 UTC www.ncbi.nlm.nih.gov [Source type: Academic]

^ Viral identification was performed using a PCR-based technique.
  • Journal of Applied Oral Science - Reverse transcription and polymerase chain reaction: principles and applications in dentistry 16 January 2010 13:52 UTC www.scielo.br [Source type: Academic]

.Some PCR 'fingerprints' methods have high discriminative power and can be used to identify genetic relationships between individuals, such as parent-child or between siblings, and are used in paternity testing (Fig.^ Some PCR fingerprints methods have high discriminative power and can be used to identify genetic relationships between individuals, such as parent-child or between siblings, and are used in paternity testing.
  • Polymerase Chain Reaction (PCR) K-12 Experiments and Background Information 16 January 2010 13:52 UTC www.juliantrubin.com [Source type: Academic]

^ Although these resulting 'fingerprints' are unique (except for identical twins), 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.
  • What Is Pcr? 17 January 2010 19:019 UTC www.bionewsonline.com [Source type: Academic]
  • Polymerase chain reaction - Biocrawler, the free encyclopedia 17 January 2010 19:019 UTC www.biocrawler.com [Source type: Academic]
  • Polymerase chain reaction - Hwiki 16 January 2010 13:52 UTC hwiki.fzk.de [Source type: Academic]

^ However, the use of PCR as a test for cure has some potential limitations.
  • Diagnosis and Monitoring of Whipple Disease by Polymerase Chain Reaction — Ann Intern Med 16 January 2010 13:52 UTC www.annals.org [Source type: Academic]

4). .This technique may also be used to determine evolutionary relationships among organisms.^ A variation of this technique can also be used to determine evolutionary relationships between organisms.
  • What Is Pcr? 17 January 2010 19:019 UTC www.bionewsonline.com [Source type: Academic]
  • Polymerase chain reaction - Biocrawler, the free encyclopedia 17 January 2010 19:019 UTC www.biocrawler.com [Source type: Academic]
  • Polymerase chain reaction - Hwiki 16 January 2010 13:52 UTC hwiki.fzk.de [Source type: Academic]

^ This technique may also be used to determine evolutionary relationships among organisms.
  • Polymerase Chain Reaction (PCR) K-12 Experiments and Background Information 16 January 2010 13:52 UTC www.juliantrubin.com [Source type: Academic]

^ An analogous method may be used in which the patient sample is PBMC, and the presence of a mutation is proviral DNA is determined.
  • Polymerase chain reaction assays for monitoring antiviral therapy and making therapeutic decisions in the treatment of acquired immunodeficiency syndrome - Patent 5650268 16 January 2010 13:52 UTC www.freepatentsonline.com [Source type: Academic]

.Figure 4: Electrophoresis of PCR-amplified DNA fragments.^ File:Pcr fingerprint.png Figure 4 : Electrophoresis of PCR-amplified DNA fragments.
  • Polymerase chain reaction - Hwiki 16 January 2010 13:52 UTC hwiki.fzk.de [Source type: Academic]

^ When multiple primer sets were used, reproducible patterns of amplified complementary DNA fragments were obtained that showed strong dependence on sequence specificity of either primer.
  • Differential display of eukaryotic messenger RNA by means of the polymerase chain reaction -- Liang and Pardee 257 (5072): 967 -- Science 16 January 2010 13:52 UTC www.sciencemag.org [Source type: Academic]

^ The size(s) of PCR products is determined by comparison with a DNA ladder (a molecular weight marker), which contains DNA fragments of known size, run on the gel alongside the PCR products.
  • Polymerase Chain Reaction (PCR) K-12 Experiments and Background Information 16 January 2010 13:52 UTC www.juliantrubin.com [Source type: Academic]

(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.

Amplification and quantification of DNA

.Because PCR amplifies the regions of DNA that it targets, PCR can be used to analyze extremely small amounts of sample.^ Because PCR amplifies the regions of DNA that it targets, PCR can be used to analyze extremely small amounts of sample.
  • Polymerase Chain Reaction (PCR) K-12 Experiments and Background Information 16 January 2010 13:52 UTC www.juliantrubin.com [Source type: Academic]

^ A 1 kb region is amplified by PCR for 4 cycles....

^ A sample of the TARGET DNA to be copied.
  • CHAPTER #10: GENETIC ENGINEERING 16 January 2010 13:52 UTC www.slic2.wsu.edu:82 [Source type: FILTERED WITH BAYES]

.This is often critical for forensic analysis, when only a trace amount of DNA is available as evidence.^ At the end of many cycles, the pool is greatly enriched in the small pieces of DNA that have the target sequences, and this amplified genetic information is then available for further analysis."

^ PCR is by far the most common method for presenting DNA evidence in a forensic context.

^ Melting curve analysis of an SNP with nearest neighbor symmetry after addition of an optimal amount of wild-type DNA and PCR amplification.
  • ScienceDirect - Analytical Biochemistry : Quantitative heteroduplex analysis for single nucleotide polymorphism genotyping 17 January 2010 19:019 UTC www.math.utah.edu [Source type: Academic]

.PCR may also be used in the analysis of ancient DNA that is tens of thousands of years old.^ Analysis of ancient DNA Using PCR, it becomes possible to analyze DNA that is thousands of years old.

^ 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.

^ In vitro AZT susceptibility testing was performed on 17 of 38 patients using a different aliquot of the same post-treatment PBMC that were used for the PCR analysis.
  • Polymerase chain reaction assays for monitoring antiviral therapy and making therapeutic decisions in the treatment of acquired immunodeficiency syndrome - Patent 5650268 16 January 2010 13:52 UTC www.freepatentsonline.com [Source type: Academic]

.These PCR-based 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.^ Viral identification was performed using a PCR-based technique.
  • Journal of Applied Oral Science - Reverse transcription and polymerase chain reaction: principles and applications in dentistry 16 January 2010 13:52 UTC www.scielo.br [Source type: Academic]

^ Half of the papers published in that year using PCR used the technique specifically for diagnostic applications.
  • Journal of Biomedical Discovery and Collaboration | Full text | The effects of business practices, licensing, and intellectual property on development and dissemination of the polymerase chain reaction: case study 16 January 2010 13:52 UTC www.j-biomed-discovery.com [Source type: Academic]

^ Using PCR, it becomes possible to analyze DNA that is thousands of years old.
  • Polymerase chain reaction - Hwiki 16 January 2010 13:52 UTC hwiki.fzk.de [Source type: Academic]

[16]
.Quantitative PCR methods allow the estimation of the amount of a given sequence present in a sample – a technique often applied to quantitatively determine levels of gene expression.^ The MSP-PCR method can be applied to any genes whose partial sequences are already known.
  • Multiplex polymerase chain reaction (PCR) with color-tagged module-shuffling primers for comparing gene expression levels in various cells -- Uematsu et al. 29 (16): e84 -- Nucleic Acids Research 16 January 2010 13:52 UTC nar.oxfordjournals.org [Source type: Academic]

^ Researchers have used traditional PCR as a way to estimate changes in the amount of a gene's expression .
  • Polymerase chain reaction - Biocrawler, the free encyclopedia 17 January 2010 19:019 UTC www.biocrawler.com [Source type: Academic]
  • Polymerase chain reaction - Hwiki 16 January 2010 13:52 UTC hwiki.fzk.de [Source type: Academic]

^ Developed in 1984 by Kary Mullis , PCR is now a common and often indispensable technique used in medical and biological research labs for a variety of applications.
  • Polymerase Chain Reaction (PCR) K-12 Experiments and Background Information 16 January 2010 13:52 UTC www.juliantrubin.com [Source type: Academic]

.Real-time PCR is an established tool for DNA quantification that measures the accumulation of DNA product after each round of PCR amplification.^ The power of real-time PCR. Adv.
  • Internal Controls for Quantitative Polymerase Chain Reaction of Swine Mammary Glands During Pregnancy and Lactation -- Tramontana et al. 91 (8): 3057 -- Journal of Dairy Science 16 January 2010 13:52 UTC jds.fass.org [Source type: Academic]

^ Recently, real-time PCR, which combines amplification and product detection in 1 step, was introduced.
  • Polymerase Chain Reaction-Based Method for Quantifying Recruitment of Monocytes to Mouse Atherosclerotic Lesions In Vivo : Enhancement by Tumor Necrosis Factor-{alpha} and Interleukin-1{beta} -- Kim et al. 20 (8): 1976 -- Arteriosclerosis, Thrombosis, and Vascular Biology 16 January 2010 13:52 UTC atvb.ahajournals.org [Source type: Academic]

^ Real-time PCR Kit .
  • PCR kits 17 January 2010 19:019 UTC www.gentaur.com [Source type: FILTERED WITH BAYES]

PCR in diagnosis of diseases

.PCR permits early diagnosis of malignant diseases such as leukemia and lymphomas, which is currently the highest developed in cancer research and is already being used routinely.^ It is expected that PCR will be useful in the microbiological diagnosis of periodontal disease 48 .
  • Journal of Applied Oral Science - Reverse transcription and polymerase chain reaction: principles and applications in dentistry 16 January 2010 13:52 UTC www.scielo.br [Source type: Academic]

^ "PCR is now being used to determine the genetic basis of complex diseases such as heart disease and diabetes," says William Nierman, director of the Rockville, MD-based American Type Culture Collection's program in molecular biology.
  • SCIENCE ELECTRONIC MARKETPLACE: STATISTICAL SOFTWARE SUPPLEMENT 17 January 2010 19:019 UTC www.sciencemag.org [Source type: FILTERED WITH BAYES]

^ Although PCR testing can detect HGV testing, such testing would not influence management because the disease is mild, and there is no known method to treat or prevent it.
  • Polymerase Chain Reaction Testing: Selected Indications 16 January 2010 13:52 UTC www.aetna.com [Source type: Academic]

.May 2008" style="white-space:nowrap;">[citation needed] PCR assays can be performed directly on genomic DNA samples to detect translocation-specific malignant cells at a sensitivity which is at least 10,000 fold higher than other methods.^ Other PCR applications capitalize on the sensitivity and specificity of the process.
  • SCIENCE ELECTRONIC MARKETPLACE: STATISTICAL SOFTWARE SUPPLEMENT 17 January 2010 19:019 UTC www.sciencemag.org [Source type: FILTERED WITH BAYES]

^ The sensitivities and specificities of antibody detection and PCR have not been determined (WHO, 1999).
  • Polymerase Chain Reaction Testing: Selected Indications 16 January 2010 13:52 UTC www.aetna.com [Source type: Academic]

^ Other methods to increase specificity include Nested PCR and Touchdown PCR .
  • Polymerase chain reaction - Hwiki 16 January 2010 13:52 UTC hwiki.fzk.de [Source type: Academic]

[citation needed]
.PCR also permits identification of non-cultivatable or slow-growing microorganisms such as mycobacteria, anaerobic bacteria, or viruses from tissue culture assays and animal models.^ The use of the method as a tool to detect microorganisms, particularly viruses and bacteria, in cutaneous tissue and its potential other future applications are described as well.
  • Journal of Investigative Dermatology - Abstract of article: Gene Amplification by Polymerase Chain Reaction in Dermatology 16 January 2010 13:52 UTC www.nature.com [Source type: Academic]

^ 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 .
  • Polymerase chain reaction - Hwiki 16 January 2010 13:52 UTC hwiki.fzk.de [Source type: Academic]

^ 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.

.The basis for PCR diagnostic applications in microbiology is the detection of infectious agents and the discrimination of non-pathogenic from pathogenic strains by virtue of specific genes.^ For detection of a determined infectious agent, specific primers for pathogen DNA sequences are chosen.
  • Journal of Applied Oral Science - Reverse transcription and polymerase chain reaction: principles and applications in dentistry 16 January 2010 13:52 UTC www.scielo.br [Source type: Academic]

^ It is only appropriate for individual tests to be selected to detect particular infectious agents if the patient's clinical presentation suggests active infection with that infectious agent.
  • Polymerase Chain Reaction Testing: Selected Indications 16 January 2010 13:52 UTC www.aetna.com [Source type: Academic]

^ Assuming that the conventional diagnostic methods represent the gold standard, the sensitivity and specificity of tracheal aspirate PCR were 79% and 96%, respectively.
  • Tracheal Aspirate as a Substrate for Polymerase Chain Reaction Detection of Viral Genome in Childhood Pneumonia and Myocarditis -- Akhtar et al. 99 (15): 2011 -- Circulation 16 January 2010 13:52 UTC circ.ahajournals.org [Source type: Academic]

[citation needed]
.Viral DNA can likewise be detected by PCR. The primers used need to be specific to the targeted sequences in the DNA of a virus, and the PCR can be used for diagnostic analyses or DNA sequencing of the viral genome.^ For detection of a determined infectious agent, specific primers for pathogen DNA sequences are chosen.
  • Journal of Applied Oral Science - Reverse transcription and polymerase chain reaction: principles and applications in dentistry 16 January 2010 13:52 UTC www.scielo.br [Source type: Academic]

^ Presentation of the target DNA, primers and probes sequences.
  • PLoS Neglected Tropical Diseases: T. cruzi OligoC-TesT: A Simplified and Standardized Polymerase Chain Reaction Format for Diagnosis of Chagas Disease 16 January 2010 13:52 UTC www.plosntds.org [Source type: Academic]

^ A universal sequencing primer can be used to sequence (more...
  • PCR, DNA sequencing and in vitro mutagenesis -- Human Molecular Genetics 2 -- NCBI Bookshelf 17 January 2010 19:019 UTC www.ncbi.nlm.nih.gov [Source type: Academic]

.The high sensitivity of PCR permits virus detection soon after infection and even before the onset of disease.^ PCR can even diagnose the diseases of the past.
  • PCR 17 January 2010 19:019 UTC www.nhlcyberfamily.org [Source type: Academic]
  • The polymerase chain reaction -- Powledge 28 (2): 44 -- Advances in Physiology Education 16 January 2010 13:52 UTC advan.physiology.org [Source type: Academic]

^ Duration of human immunodeficiency virus infection before detection of antibody.
  • Polymerase Chain Reaction for the Diagnosis of HIV Infection in Adults — Ann Intern Med 16 January 2010 13:52 UTC www.annals.org [Source type: Academic]

^ Our proprietary miRNA detection technologies enable uniformly high PCR amplification efficiencies, allowing simultaneous detection of miRNA under uniform cycling conditions.
  • Genome-Wide or Pathway-Focused miRNA Profiling 17 January 2010 19:019 UTC www.sabiosciences.com [Source type: Academic]

.Such early detection may give physicians a significant lead in treatment.^ Such early diagnoses give physicians a significant lead in treatment.
  • What Is Pcr? 17 January 2010 19:019 UTC www.bionewsonline.com [Source type: Academic]
  • Polymerase chain reaction - Biocrawler, the free encyclopedia 17 January 2010 19:019 UTC www.biocrawler.com [Source type: Academic]
  • Polymerase chain reaction - Hwiki 16 January 2010 13:52 UTC hwiki.fzk.de [Source type: Academic]

^ Such early detection may give physicians a significant lead in treatment.
  • Polymerase Chain Reaction (PCR) K-12 Experiments and Background Information 16 January 2010 13:52 UTC www.juliantrubin.com [Source type: Academic]

^ Genital infections caused by C. trachomatis are often asymptomaticz and early detection to avoid serious complications is of real value since effective treatment exists.
  • Use of polymerase chain reaction (PCR) for detection of Chlamydia trachomatis infection in cervical swab samples Jayanti Mania-Pramanik, UM Donde, Anurupa Maitra - Indian J Dermatol Venereol Leprol 16 January 2010 13:52 UTC www.ijdvl.com [Source type: Academic]

.The amount of virus ("viral load") in a patient can also be quantified by PCR-based DNA quantitation techniques (see below).^ Standard PCR-based DNA labeling .
  • PCR, DNA sequencing and in vitro mutagenesis -- Human Molecular Genetics 2 -- NCBI Bookshelf 17 January 2010 19:019 UTC www.ncbi.nlm.nih.gov [Source type: Academic]

^ Viral identification was performed using a PCR-based technique.
  • Journal of Applied Oral Science - Reverse transcription and polymerase chain reaction: principles and applications in dentistry 16 January 2010 13:52 UTC www.scielo.br [Source type: Academic]

^ Human immunodeficiency virus type 1-specific cytotoxic T lymphocyte activity is inversely correlated with HIV type 1 viral load in HIV type 1-infected long-term survivors.
  • Concerns about HIV/AIDS Testing and Measurement 16 January 2010 13:52 UTC rethinkaids.com [Source type: Academic]

Variations on the basic PCR technique

.
  • Allele-specific PCR: a diagnostic or cloning technique which is based on single-nucleotide polymorphisms (SNPs) (single-base differences in DNA).^ Allele-specific PCR (ARMS test) .
    • PCR, DNA sequencing and in vitro mutagenesis -- Human Molecular Genetics 2 -- NCBI Bookshelf 17 January 2010 19:019 UTC www.ncbi.nlm.nih.gov [Source type: Academic]

    ^ Standard PCR-based DNA labeling .
    • PCR, DNA sequencing and in vitro mutagenesis -- Human Molecular Genetics 2 -- NCBI Bookshelf 17 January 2010 19:019 UTC www.ncbi.nlm.nih.gov [Source type: Academic]

    ^ In this way, PCR is used in detection assays for infectious agents, pre-natal diagnosis of genetic disease, genomic DNA or cDNA cloning, rare RNA quantification, RNA amplification by RT-PCR, in vitro mutagenesis and recombinant DNA manufacture, studies of forensic samples, and variation analysis of alleles sequences.
    • Journal of Applied Oral Science - Reverse transcription and polymerase chain reaction: principles and applications in dentistry 16 January 2010 13:52 UTC www.scielo.br [Source type: Academic]

    .It requires prior knowledge of a DNA sequence, including differences between alleles, and uses primers whose 3' ends encompass the SNP. PCR amplification under stringent conditions is much less efficient in the presence of a mismatch between template and primer, so successful amplification with an SNP-specific primer signals presence of the specific SNP in a sequence.^ The probe hybridizes specifically with the target sequence at a position between the PCR primers.
    • Analysis of Specific Bacteria from Environmental Samples using a Quantitative Polymerase Chain Reaction 16 January 2010 13:52 UTC www.open-access-biology.com [Source type: Academic]

    ^ Design and analyze primers for different types of PCR-based amplifications.
    • HSLS: PCR primers, oligos databases and design tools 17 January 2010 19:019 UTC www.hsls.pitt.edu [Source type: Academic]

    ^ Amplification of midivariant DNA templates .
    • Coupled polymerase chain reaction ... - Google Patent Search 16 January 2010 13:52 UTC www.google.com [Source type: Reference]

    [17] .See SNP genotyping for more information.
  • Assembly PCR or Polymerase Cycling Assembly (PCA): artificial synthesis of long DNA sequences by performing PCR on a pool of long oligonucleotides with short overlapping segments.^ 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.
    • Polymerase chain reaction - Hwiki 16 January 2010 13:52 UTC hwiki.fzk.de [Source type: Academic]

    ^ Need for target DNA sequence information .
    • PCR, DNA sequencing and in vitro mutagenesis -- Human Molecular Genetics 2 -- NCBI Bookshelf 17 January 2010 19:019 UTC www.ncbi.nlm.nih.gov [Source type: Academic]

    ^ Second, the PCR primers, which are short pieces of DNA (oligonucleotides) 20-30 base pairs in length exactly complementary to the ends of each piece of the double-stranded DNA to be amplified, anneal to their complementary regions of the DNA. Third, synthesis of the complementary strand of DNA occurs in the presence of the enzyme Taq polymerase and nucleotides triphosphates (dATP, dCTP, dGTP, and dTTP).
    • Polymerase Chain Reaction Testing: Selected Indications 16 January 2010 13:52 UTC www.aetna.com [Source type: Academic]

    .The oligonucleotides alternate between sense and antisense directions, and the overlapping segments determine the order of the PCR fragments, thereby selectively producing the final long DNA product.^ Only the reaction between MSPs and corresponding DNA fragments produced PCR products.
    • Multiplex polymerase chain reaction (PCR) with color-tagged module-shuffling primers for comparing gene expression levels in various cells -- Uematsu et al. 29 (16): e84 -- Nucleic Acids Research 16 January 2010 13:52 UTC nar.oxfordjournals.org [Source type: Academic]

    ^ Primers The DNA fragment to be amplified is determined by selecting primers.

    ^ 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.
    • Polymerase chain reaction - Hwiki 16 January 2010 13:52 UTC hwiki.fzk.de [Source type: Academic]

    [18]
  • .
  • Asymmetric PCR: preferentially amplifies one DNA strand in a double-stranded DNA template.^ The beauty of PCR is it is so powerful that one can amplify from a single DNA molecule.
    • UNIT 8: POLYMERASE CHAIN REACTION (PCR) 16 January 2010 13:52 UTC www.med.unc.edu [Source type: Academic]

    ^ Asymmetric PCR - Asymmetric PCR is used to preferentially amplify one strand of the original DNA more than the other.
    • Polymerase chain reaction - Hwiki 16 January 2010 13:52 UTC hwiki.fzk.de [Source type: Academic]

    ^ RNA, or ribonucleic acid , is very similar to DNA except that it is happy to live in a single-stranded state (as opposed to DNA's desire to form complementary double-stranded helixes).

    .It is used in sequencing and hybridization probing where amplification of only one of the two complementary strands is required.^ It finds use in some types of sequencing and hybridization probing where having only one of the two complementary stands is ideal.
    • Polymerase chain reaction - Hwiki 16 January 2010 13:52 UTC hwiki.fzk.de [Source type: Academic]

    ^ Primers used for amplification and sequencing .
    • PLoS ONE: Evolution in Quantum Leaps: Multiple Combinatorial Transfers of HPI and Other Genetic Modules in Enterobacteriaceae 17 January 2010 19:019 UTC www.plosone.org [Source type: Academic]

    ^ It is feasible to use the RealMaster Probe kits with these platforms; however, optimization work will be required.
    • Brinkmann Instruments Canada ::: FAQs-PCR 17 January 2010 19:019 UTC www.brinkmanncanada.com [Source type: FILTERED WITH BAYES]

    .PCR is carried out as usual, but with a great excess of the primer for the strand targeted for amplification.^ PCR is carried out as usual, but with a great excess of the primers for the chosen strand.
    • Polymerase chain reaction - Hwiki 16 January 2010 13:52 UTC hwiki.fzk.de [Source type: Academic]

    ^ Multiplex quantitative PCR amplification of Tg using at a variety of different primer pairs, as well as a control sequence, may provide the most clinical information.
    • Quantitative Reverse Transcription-Polymerase Chain Reaction of Circulating Thyroglobulin Messenger Ribonucleic Acid for Monitoring Patients with Thyroid Carcinoma -- Ringel et al. 84 (11): 4037 -- Journal of Clinical Endocrinology & Metabolism 16 January 2010 13:52 UTC jcem.endojournals.org [Source type: Academic]

    ^ For one thing, the crude PCR mix contains a large excess of primers and buffer salts that can interfere with sequencing.
    • UNIT 8: POLYMERASE CHAIN REACTION (PCR) 16 January 2010 13:52 UTC www.med.unc.edu [Source type: Academic]

    .Because of the slow (arithmetic) amplification later in the reaction after the limiting primer has been used up, extra cycles of PCR are required.^ Use 10 m l for 50 m l PCR reaction.
    • UNIT 8: POLYMERASE CHAIN REACTION (PCR) 16 January 2010 13:52 UTC www.med.unc.edu [Source type: Academic]

    ^ The more initial templates there are, the more nucleotides are used up initially, which limits the efficiency of amplifications in the later cycles.
    • UNIT 8: POLYMERASE CHAIN REACTION (PCR) 16 January 2010 13:52 UTC www.med.unc.edu [Source type: Academic]

    ^ Use of degenerate primers can greatly reduce the specificity of the PCR amplification.
    • Polymerase chain reaction - Hwiki 16 January 2010 13:52 UTC hwiki.fzk.de [Source type: Academic]

    [19] .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.^ A recent modification on this process, known as L inear- A fter- T he- E xponential-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.
    • Polymerase chain reaction - Hwiki 16 January 2010 13:52 UTC hwiki.fzk.de [Source type: Academic]

    ^ Use 10 m l for 50 m l PCR reaction.
    • UNIT 8: POLYMERASE CHAIN REACTION (PCR) 16 January 2010 13:52 UTC www.med.unc.edu [Source type: Academic]

    ^ The melting point of the primer sets the upper limit on annealing temperature.

    [20]
  • .
  • Helicase-dependent amplification: similar to traditional PCR, but uses a constant temperature rather than cycling through denaturation and annealing/extension cycles.^ PCR mixture through PCR temperatures; and .
    • Polymerase chain reaction container and methods of using the same - Patent 5576197 16 January 2010 13:52 UTC www.freepatentsonline.com [Source type: Reference]

    ^ Recent developments in PCR techniques A more recent method which excludes a temperature cycle, but uses enzymes, is helicase-dependent amplification.

    ^ A more recent method which excludes a temperature cycle, but uses enzymes, is helicase-dependent amplification .
    • Polymerase chain reaction - Hwiki 16 January 2010 13:52 UTC hwiki.fzk.de [Source type: Academic]

    .DNA helicase, an enzyme that unwinds DNA, is used in place of thermal denaturation.^ A helicase, which unwinds the DNA ahead of the fork.

    ^ The PCR process involved heating the DNA to 95 degrees Celsius, which had the effect of denaturing the E. coli polymerase enzyme they were using, rendering it inactive for the next round of amplification.
    • Journal of Biomedical Discovery and Collaboration | Full text | The effects of business practices, licensing, and intellectual property on development and dissemination of the polymerase chain reaction: case study 16 January 2010 13:52 UTC www.j-biomed-discovery.com [Source type: Academic]

    ^ File:Pcr machine.jpg Figure 1 : A thermal cycler for PCR PCR is used to amplify specific regions of a DNA strand.
    • Polymerase chain reaction - Hwiki 16 January 2010 13:52 UTC hwiki.fzk.de [Source type: Academic]

    [21]
  • .
  • Hot-start PCR: a technique that reduces non-specific amplification during the initial set up stages of the PCR. It may be performed manually by heating the reaction components to the melting temperature (e.g., 95°C) before adding the polymerase.^ Polymerase chain reaction (PCR) in various stages of HIV infection.
    • Polymerase Chain Reaction for the Diagnosis of HIV Infection in Adults — Ann Intern Med 16 January 2010 13:52 UTC www.annals.org [Source type: Academic]

    ^ In hot start PCR, a PCR mixture lacking a critical reagent(s) is heated to the polymerase chain reaction temperature and, at that temperature, the missing critical reagent(s) is added to the PCR mixture.
    • Polymerase chain reaction container and methods of using the same - Patent 5576197 16 January 2010 13:52 UTC www.freepatentsonline.com [Source type: Reference]

    ^ PCR may then be performed as follows.
    • Polymerase chain reaction assays for monitoring antiviral therapy and making therapeutic decisions in the treatment of acquired immunodeficiency syndrome - Patent 5650268 16 January 2010 13:52 UTC www.freepatentsonline.com [Source type: Academic]

    [22] .Specialized enzyme systems have been developed that inhibit the polymerase's activity at ambient temperature, either by the binding of an antibody[9] or by the presence of covalently bound inhibitors that only dissociate after a high-temperature activation step.^ Specialized enzyme systems have been developed that inhibit the polymerase's activity at ambient temperature, either by the binding of an antibody or by the presence of covalently bound inhibitors that only dissociate after a high-temperature activation step.
    • Polymerase chain reaction - Hwiki 16 January 2010 13:52 UTC hwiki.fzk.de [Source type: Academic]

    ^ A specially engineered TopoTaq polymerase activates instantly at high temperature and overcomes limitations of conventional "hot-start" enzymes that have to wait for an antibody to dislodge while target DNA degrades at 95˚C. In addition, its activity is blocked upon completion of PCR at low temperature.
    • Polymerase chain reaction - Hwiki 16 January 2010 13:52 UTC hwiki.fzk.de [Source type: Academic]

    ^ Non-specific priming can be prevented during the low temperatures of reaction preparation by use of "hot-start" polymerase enzymes where the active site is blocked by an antibody or chemical that only dislodges once the reaction is heated to 95˚C during the denaturation step of the first cycle.
    • Polymerase chain reaction - Hwiki 16 January 2010 13:52 UTC hwiki.fzk.de [Source type: Academic]

    .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 PCR (ISSR): a PCR method for DNA fingerprinting that amplifies regions between simple sequence repeats to produce a unique fingerprint of amplified fragment lengths.^ Hot-start/cold-finish PCR is achieved with new hybrid polymerases that are inactive at ambient temperature and are instantly activated at elongation temperature.
    • Polymerase chain reaction - Hwiki 16 January 2010 13:52 UTC hwiki.fzk.de [Source type: Academic]

    ^ PCR is the enzymatic amplification of a specific DNA sequence in vitro 9 .
    • Journal of Applied Oral Science - Reverse transcription and polymerase chain reaction: principles and applications in dentistry 16 January 2010 13:52 UTC www.scielo.br [Source type: Academic]

    ^ Hot-start PCR .
    • PCR, DNA sequencing and in vitro mutagenesis -- Human Molecular Genetics 2 -- NCBI Bookshelf 17 January 2010 19:019 UTC www.ncbi.nlm.nih.gov [Source type: Academic]

    [23]
  • .
  • Inverse PCR: is commonly used to identify the flanking sequences around genomic inserts.^ This is especially useful in identifying flanking sequences to various genomic inserts.
    • Polymerase chain reaction - Hwiki 16 January 2010 13:52 UTC hwiki.fzk.de [Source type: Academic]

    ^ Multiplex quantitative PCR amplification of Tg using at a variety of different primer pairs, as well as a control sequence, may provide the most clinical information.
    • Quantitative Reverse Transcription-Polymerase Chain Reaction of Circulating Thyroglobulin Messenger Ribonucleic Acid for Monitoring Patients with Thyroid Carcinoma -- Ringel et al. 84 (11): 4037 -- Journal of Clinical Endocrinology & Metabolism 16 January 2010 13:52 UTC jcem.endojournals.org [Source type: Academic]

    ^ These are known, absolutely identified to be, the sequence of letters which ONLY flank a particular region of a particular organism's DNA, and NO OTHER ORGANISM'S DNA. This region would be a target sequence for PCR. .

    .It involves a series of DNA digestions and self ligation, resulting in known sequences at either end of the unknown sequence.^ This involves a series of digestions and self ligation before cutting by an endonuclease , resulting in known sequences at either end of the unknown sequence.
    • Polymerase chain reaction - Hwiki 16 January 2010 13:52 UTC hwiki.fzk.de [Source type: Academic]

    ^ When multiple primer sets were used, reproducible patterns of amplified complementary DNA fragments were obtained that showed strong dependence on sequence specificity of either primer.
    • Differential display of eukaryotic messenger RNA by means of the polymerase chain reaction -- Liang and Pardee 257 (5072): 967 -- Science 16 January 2010 13:52 UTC www.sciencemag.org [Source type: Academic]

    ^ For example, the sequence of base pairs GAATTC is a palindrome because both sequences of the double strand READ THE SAME when read from either their respective "G" or "C" ends ( COMPLEMENTARY strand = CTTAAG).
    • CHAPTER #10: GENETIC ENGINEERING 16 January 2010 13:52 UTC www.slic2.wsu.edu:82 [Source type: FILTERED WITH BAYES]

    [24]
  • .
  • Ligation-mediated PCR: uses small DNA linkers ligated to the DNA of interest and multiple primers annealing to the DNA linkers; it has been used for DNA sequencing, genome walking, and DNA footprinting.^ Linker-primed PCR (ligation adaptor PCR) .
    • PCR, DNA sequencing and in vitro mutagenesis -- Human Molecular Genetics 2 -- NCBI Bookshelf 17 January 2010 19:019 UTC www.ncbi.nlm.nih.gov [Source type: Academic]

    ^ Automated DNA sequencing using fluorescent primers Figure 6.17 .
    • PCR, DNA sequencing and in vitro mutagenesis -- Human Molecular Genetics 2 -- NCBI Bookshelf 17 January 2010 19:019 UTC www.ncbi.nlm.nih.gov [Source type: Academic]

    ^ PCR-amplified products are often used for DNA sequencing .
    • PCR, DNA sequencing and in vitro mutagenesis -- Human Molecular Genetics 2 -- NCBI Bookshelf 17 January 2010 19:019 UTC www.ncbi.nlm.nih.gov [Source type: Academic]

    [25]
  • .
  • Methylation-specific PCR (MSP): developed by Stephen Baylin and Jim Herman at the Johns Hopkins School of Medicine,[26] 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.^ Species-specific ribosomal 16S primers for PCR amplification were developed for detection of new species.
    • Journal of Applied Oral Science - Reverse transcription and polymerase chain reaction: principles and applications in dentistry 16 January 2010 13:52 UTC www.scielo.br [Source type: Academic]

    ^ Standard PCR-based DNA labeling .
    • PCR, DNA sequencing and in vitro mutagenesis -- Human Molecular Genetics 2 -- NCBI Bookshelf 17 January 2010 19:019 UTC www.ncbi.nlm.nih.gov [Source type: Academic]

    ^ In DNA, adenine and thymine, as well as guanine and cytosine, can be a base pair.

    .Two PCRs are then carried out on the modified DNA, using primer sets identical except at any CpG islands within the primer sequences.^ Two PCR reactions are then carried out on the modified DNA, using primer sets identical except at any CpG islands within the primer sequences.
    • Polymerase chain reaction - Hwiki 16 January 2010 13:52 UTC hwiki.fzk.de [Source type: Academic]

    ^ 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.
    • Polymerase chain reaction - Hwiki 16 January 2010 13:52 UTC hwiki.fzk.de [Source type: Academic]

    ^ The TaqMan™ 5′ exonuclease assay In addition to two conventional PCR primers, P1 and P2, which are specific for the target sequence, a third primer , P3, is designed to bind specifically to a site on the target sequence downstream of the P1 binding site.
    • PCR, DNA sequencing and in vitro mutagenesis -- Human Molecular Genetics 2 -- NCBI Bookshelf 17 January 2010 19:019 UTC www.ncbi.nlm.nih.gov [Source type: Academic]

    .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.
  • Miniprimer PCR: uses a thermostable polymerase (S-Tbr) that can extend from short primers ("smalligos") as short as 9 or 10 nucleotides.^ 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.
    • Polymerase chain reaction - Hwiki 16 January 2010 13:52 UTC hwiki.fzk.de [Source type: Academic]

    ^ The DNA in the nuclei of these cells is amplified using the PCR technique (polymerase chain reaction).

    ^ PCR is used to amplify a short, well-defined part of a DNA strand.

    .This method permits PCR targeting to smaller primer binding regions, and is used to amplify conserved DNA sequences, such as the 16S (or eukaryotic 18S) rRNA gene.^ PCR is an in vitro method for amplifying DNA sequences using defined oligonucleotide primers Oligonucleotide primers A and B are complementary to DNA sequences located on opposite DNA strands and flanking the region to be amplified.
    • PCR, DNA sequencing and in vitro mutagenesis -- Human Molecular Genetics 2 -- NCBI Bookshelf 17 January 2010 19:019 UTC www.ncbi.nlm.nih.gov [Source type: Academic]

    ^ Presentation of the target DNA, primers and probes sequences.
    • PLoS Neglected Tropical Diseases: T. cruzi OligoC-TesT: A Simplified and Standardized Polymerase Chain Reaction Format for Diagnosis of Chagas Disease 16 January 2010 13:52 UTC www.plosntds.org [Source type: Academic]

    ^ Need for target DNA sequence information .
    • PCR, DNA sequencing and in vitro mutagenesis -- Human Molecular Genetics 2 -- NCBI Bookshelf 17 January 2010 19:019 UTC www.ncbi.nlm.nih.gov [Source type: Academic]

    .[27]
  • Multiplex-PCR: consists of multiple primer sets within a single PCR mixture to produce amplicons of varying sizes that are specific to different DNA sequences.^ Presentation of the target DNA, primers and probes sequences.
    • PLoS Neglected Tropical Diseases: T. cruzi OligoC-TesT: A Simplified and Standardized Polymerase Chain Reaction Format for Diagnosis of Chagas Disease 16 January 2010 13:52 UTC www.plosntds.org [Source type: Academic]

    ^ PCR is the enzymatic amplification of a specific DNA sequence in vitro 9 .
    • Journal of Applied Oral Science - Reverse transcription and polymerase chain reaction: principles and applications in dentistry 16 January 2010 13:52 UTC www.scielo.br [Source type: Academic]

    ^ The complete gene was amplified by two sets of specific primers.
    • PLoS ONE: Evolution in Quantum Leaps: Multiple Combinatorial Transfers of HPI and Other Genetic Modules in Enterobacteriaceae 17 January 2010 19:019 UTC www.plosone.org [Source type: Academic]

    .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.^ More appropriate clinical information may be gained from quantitative studies.
    • HMDS Techniques: Molecular Diagnosis by Polymerase Chain Reaction 16 January 2010 13:52 UTC www.hmds.org.uk [Source type: Academic]

    ^ By targeting multiple genes at once, additional information may be elicited from a single test run that otherwise would require several times the reagents and technician time to perform.
    • Polymerase chain reaction - Hwiki 16 January 2010 13:52 UTC hwiki.fzk.de [Source type: Academic]

    ^ In addition, as skilled labour is required to handle the reagents with extreme care, laboratories will need to ensure that their employees are well trained in the modus operandi of the procedures.
    • European PCR Reagents Markets for Clinical Applications 17 January 2010 19:019 UTC www.frost.com [Source type: Academic]

    .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: increases the specificity of DNA amplification, by reducing background due to non-specific amplification of DNA. Two sets of primers are used in two successive PCRs.^ Agarose gel electrophoresis is used to visualize the PCR products.
    • Methylation Promoter PCR Kit 17 January 2010 19:019 UTC www.panomics.com [Source type: Academic]

    ^ Use 10 m l for 50 m l PCR reaction.
    • UNIT 8: POLYMERASE CHAIN REACTION (PCR) 16 January 2010 13:52 UTC www.med.unc.edu [Source type: Academic]

    ^ PCR is the enzymatic amplification of a specific DNA sequence in vitro 9 .
    • Journal of Applied Oral Science - Reverse transcription and polymerase chain reaction: principles and applications in dentistry 16 January 2010 13:52 UTC www.scielo.br [Source type: Academic]

    .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 use of gene cloning to amplify the cloned DNA and/or to produce lots of the cloned gene's product.
    • CHAPTER #10: GENETIC ENGINEERING 16 January 2010 13:52 UTC www.slic2.wsu.edu:82 [Source type: FILTERED WITH BAYES]

    ^ The DNA fragment to be amplified is determined by selecting primers.
    • Polymerase chain reaction - Biocrawler, the free encyclopedia 17 January 2010 19:019 UTC www.biocrawler.com [Source type: Academic]

    ^ Higher primer concentrations may promote mispriming and accumulation of non specific product.
    • PCR (Polymerase Chain Reaction) 16 January 2010 13:52 UTC www.slideshare.net [Source type: Reference]

    .The product(s) are then used in a second PCR with a set of primers whose binding sites are completely or partially different from and located 3' of each of the primers used in the first reaction.^ Use 10 m l for 50 m l PCR reaction.
    • UNIT 8: POLYMERASE CHAIN REACTION (PCR) 16 January 2010 13:52 UTC www.med.unc.edu [Source type: Academic]

    ^ Two sets of primers are used in two successive PCR runs, the second set intended to amplify a secondary target within the first run product.
    • Polymerase chain reaction - Hwiki 16 January 2010 13:52 UTC hwiki.fzk.de [Source type: Academic]

    ^ Finally, a completely different approach to cloning PCR products is to introduce restriction sites into the 5’ end of the PCR primers used for amplification.
    • UNIT 8: POLYMERASE CHAIN REACTION (PCR) 16 January 2010 13:52 UTC www.med.unc.edu [Source type: Academic]

    .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): used to measure the quantity of a PCR product (commonly in real-time).^ The use of gene cloning to amplify the cloned DNA and/or to produce lots of the cloned gene's product.
    • CHAPTER #10: GENETIC ENGINEERING 16 January 2010 13:52 UTC www.slic2.wsu.edu:82 [Source type: FILTERED WITH BAYES]

    ^ Presentation of the target DNA, primers and probes sequences.
    • PLoS Neglected Tropical Diseases: T. cruzi OligoC-TesT: A Simplified and Standardized Polymerase Chain Reaction Format for Diagnosis of Chagas Disease 16 January 2010 13:52 UTC www.plosntds.org [Source type: Academic]

    ^ PCR is the enzymatic amplification of a specific DNA sequence in vitro 9 .
    • Journal of Applied Oral Science - Reverse transcription and polymerase chain reaction: principles and applications in dentistry 16 January 2010 13:52 UTC www.scielo.br [Source type: Academic]

    .It quantitatively measures 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.^ Quantitative PCR - Q-PCR ( Q uantitative PCR) is 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 determining whether a sequence is present or not, and if it is present the number of copies in the sample.
    • Polymerase chain reaction - Hwiki 16 January 2010 13:52 UTC hwiki.fzk.de [Source type: Academic]

    ^ A sample of the TARGET DNA to be copied.
    • CHAPTER #10: GENETIC ENGINEERING 16 January 2010 13:52 UTC www.slic2.wsu.edu:82 [Source type: FILTERED WITH BAYES]

    ^ A commonly used procedure is the "hot start".
    • UNIT 8: POLYMERASE CHAIN REACTION (PCR) 16 January 2010 13:52 UTC www.med.unc.edu [Source type: Academic]

    .Quantitative real-time PCR has a very high degree of precision.^ Quantitative real time PCR was performed by Reis, et al.
    • Journal of Applied Oral Science - Reverse transcription and polymerase chain reaction: principles and applications in dentistry 16 January 2010 13:52 UTC www.scielo.br [Source type: Academic]

    ^ Quantitative real-time PCR is often confusingly known as RT-PCR ( R eal T ime PCR) and RQ-PCR. QRT-PCR or RTQ-PCR are more appropriate contractions.
    • Polymerase chain reaction - Hwiki 16 January 2010 13:52 UTC hwiki.fzk.de [Source type: Academic]

    ^ The availability of real-time PCR makes an important difference, reducing time and energy input markedly.
    • Polymerase Chain Reaction-Based Method for Quantifying Recruitment of Monocytes to Mouse Atherosclerotic Lesions In Vivo : Enhancement by Tumor Necrosis Factor-{alpha} and Interleukin-1{beta} -- Kim et al. 20 (8): 1976 -- Arteriosclerosis, Thrombosis, and Vascular Biology 16 January 2010 13:52 UTC atvb.ahajournals.org [Source type: Academic]

    .QRT-PCR methods use fluorescent dyes, such as Sybr Green, EvaGreen or fluorophore-containing DNA probes, such as TaqMan, to measure the amount of amplified product in real time.^ The use of gene cloning to amplify the cloned DNA and/or to produce lots of the cloned gene's product.
    • CHAPTER #10: GENETIC ENGINEERING 16 January 2010 13:52 UTC www.slic2.wsu.edu:82 [Source type: FILTERED WITH BAYES]

    ^ A quantative RT-PCR method has been developed, it is called Real-time PCR .
    • Polymerase chain reaction - Hwiki 16 January 2010 13:52 UTC hwiki.fzk.de [Source type: Academic]

    ^ Real-time PCR Kit .
    • PCR kits 17 January 2010 19:019 UTC www.gentaur.com [Source type: FILTERED WITH BAYES]

    .It is also sometimes abbreviated to RT-PCR (Real Time PCR) or RQ-PCR. QRT-PCR or RTQ-PCR are more appropriate contractions, since RT-PCR commonly refers to reverse transcription PCR (see below), often used in conjunction with Q-PCR.
  • Reverse Transcription PCR (RT-PCR): for amplifying DNA from RNA. Reverse transcriptase reverse transcribes RNA into cDNA, which is then amplified by PCR. 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.^ The use of gene cloning to amplify the cloned DNA and/or to produce lots of the cloned gene's product.
    • CHAPTER #10: GENETIC ENGINEERING 16 January 2010 13:52 UTC www.slic2.wsu.edu:82 [Source type: FILTERED WITH BAYES]

    ^ What reverse transcriptase does the cMaster RT use?
    • Brinkmann Instruments Canada ::: FAQs-PCR 17 January 2010 19:019 UTC www.brinkmanncanada.com [Source type: FILTERED WITH BAYES]

    ^ Once RNA is isolated it can be reverse transcribed back into DNA (complementary DNA to be precise, known as cDNA), at which point traditional PCR can be applied to amplify the gene.

    .If the genomic DNA sequence of a gene is known, RT-PCR can be used to map the location of exons and introns in the gene.^ Quantitative real-time PCR is often confusingly known as RT-PCR ( R eal T ime PCR) and RQ-PCR. QRT-PCR or RTQ-PCR are more appropriate contractions.
    • Polymerase chain reaction - Hwiki 16 January 2010 13:52 UTC hwiki.fzk.de [Source type: Academic]

    ^ In this way, PCR is used in detection assays for infectious agents, pre-natal diagnosis of genetic disease, genomic DNA or cDNA cloning, rare RNA quantification, RNA amplification by RT-PCR, in vitro mutagenesis and recombinant DNA manufacture, studies of forensic samples, and variation analysis of alleles sequences.
    • Journal of Applied Oral Science - Reverse transcription and polymerase chain reaction: principles and applications in dentistry 16 January 2010 13:52 UTC www.scielo.br [Source type: Academic]

    ^ The presence of the 17 different genes known to be located on HPI-ICE Ec1 was assessed in 44 irp2 -positive isolates.
    • PLoS ONE: Evolution in Quantum Leaps: Multiple Combinatorial Transfers of HPI and Other Genetic Modules in Enterobacteriaceae 17 January 2010 19:019 UTC www.plosone.org [Source type: Academic]

    .The 5' end of a gene (corresponding to the transcription start site) is typically identified by RACE-PCR (Rapid Amplification of cDNA Ends).
  • Solid Phase PCR: encompasses multiple meanings, including Polony Amplification (where PCR colonies are derived in a gel matrix, for example), 'Bridge PCR' (primers are covalently linked to a solid-support surface), conventional Solid Phase PCR (where Asymmetric PCR is applied in the presence of solid support bearing primer with sequence matching one of the aqueous primers) and Enhanced Solid Phase PCR[28] (where conventional Solid Phase PCR can be improved by employing high Tm and nested solid support primer with optional application of a thermal 'step' to favour solid support priming).
  • Thermal asymmetric interlaced PCR (TAIL-PCR): for isolation of an unknown sequence flanking a known sequence.^ RACE-PCR - Rapid amplification of cDNA ends.
    • Polymerase chain reaction - Hwiki 16 January 2010 13:52 UTC hwiki.fzk.de [Source type: Academic]

    ^ Primers for PCR of the Sry gene were based on the published sequence.
    • Polymerase Chain Reaction-Based Method for Quantifying Recruitment of Monocytes to Mouse Atherosclerotic Lesions In Vivo : Enhancement by Tumor Necrosis Factor-{alpha} and Interleukin-1{beta} -- Kim et al. 20 (8): 1976 -- Arteriosclerosis, Thrombosis, and Vascular Biology 16 January 2010 13:52 UTC atvb.ahajournals.org [Source type: Academic]

    ^ A recent modification on this process, known as L inear- A fter- T he- E xponential-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.
    • Polymerase chain reaction - Hwiki 16 January 2010 13:52 UTC hwiki.fzk.de [Source type: Academic]

    .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.^ This is increased by the use of degenerate sequences or bases in the primer.
    • Polymerase chain reaction - Hwiki 16 January 2010 13:52 UTC hwiki.fzk.de [Source type: Academic]

    ^ Sometimes degenerate primers are used.

    ^ The other use for degenerate primers is when primer design is based on protein sequence.

    [29]
  • .
  • Touchdown PCR (Step-down PCR): a variant of PCR that aims to reduce nonspecific background by gradually lowering the annealing temperature as PCR cycling progresses.^ Touchdown PCR - Touchdown PCR is a variant of PCR that reduces nonspecific primer annealing by more gradually lowering the annealing temperature between cycles.
    • Polymerase chain reaction - Hwiki 16 January 2010 13:52 UTC hwiki.fzk.de [Source type: Academic]

    ^ A style of PCR that reduces nonspecific primer annealing is Touchdown PCR. .

    ^ The earliest steps of a Touchdown PCR cycle have high annealing temperatures.

    .The annealing temperature at the initial cycles is usually a few degrees (3-5°C) above the Tm of the primers used, while at the later cycles, it is a few degrees (3-5°C) below the primer Tm.^ For every subsequent cycle, the annealing temperature is decreased by 1 degree Celsius.

    ^ The calculated T m for both primers used in reaction should not differ > 5°C. The ideal annealing temperature usually is 5°C below the calculated primer T m .
    • Polymerase chain reaction - Hwiki 16 January 2010 13:52 UTC hwiki.fzk.de [Source type: Academic]

    ^ The temperature at which primers anneal during a cycle of PCR determines the specificity of annealing.

    .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.^ Specificity of amplification and primer design .
    • PCR, DNA sequencing and in vitro mutagenesis -- Human Molecular Genetics 2 -- NCBI Bookshelf 17 January 2010 19:019 UTC www.ncbi.nlm.nih.gov [Source type: Academic]

    ^ As higher temperatures give greater specificity for primer binding, primers anneal first as the temperature passes through the zone of greatest specificity.
    • Polymerase chain reaction - Hwiki 16 January 2010 13:52 UTC hwiki.fzk.de [Source type: Academic]

    ^ At lower temperatures, the primers bind less specifically.

    [30]
  • .
  • PAN-AC: uses isothermal conditions for amplification, and may be used in living cells.^ Multiplex quantitative PCR amplification of Tg using at a variety of different primer pairs, as well as a control sequence, may provide the most clinical information.
    • Quantitative Reverse Transcription-Polymerase Chain Reaction of Circulating Thyroglobulin Messenger Ribonucleic Acid for Monitoring Patients with Thyroid Carcinoma -- Ringel et al. 84 (11): 4037 -- Journal of Clinical Endocrinology & Metabolism 16 January 2010 13:52 UTC jcem.endojournals.org [Source type: Academic]

    ^ It is sometimes necessary to optimize the concentration of magnesium ions for PCR. The reaction conditions may differ with template and primers used.
    • Brinkmann Instruments Canada ::: FAQs-PCR 17 January 2010 19:019 UTC www.brinkmanncanada.com [Source type: FILTERED WITH BAYES]

    ^ Identification of circulating thyroid cells using RT-PCR amplification of thyroid-specific messenger RNA (mRNA) transcripts represents a promising alternative assay system.
    • Quantitative Reverse Transcription-Polymerase Chain Reaction of Circulating Thyroglobulin Messenger Ribonucleic Acid for Monitoring Patients with Thyroid Carcinoma -- Ringel et al. 84 (11): 4037 -- Journal of Clinical Endocrinology & Metabolism 16 January 2010 13:52 UTC jcem.endojournals.org [Source type: Academic]

    [31][32]
  • .
  • Universal Fast Walking: for genome walking and genetic fingerprinting using a more specific 'two-sided' PCR than conventional 'one-sided' approaches (using only one gene-specific primer and one general primer - which can lead to artefactual 'noise')[33] by virtue of a mechanism involving lariat structure formation.^ A universal sequencing primer can be used to sequence (more...
    • PCR, DNA sequencing and in vitro mutagenesis -- Human Molecular Genetics 2 -- NCBI Bookshelf 17 January 2010 19:019 UTC www.ncbi.nlm.nih.gov [Source type: Academic]

    ^ The complete gene was amplified by two sets of specific primers.
    • PLoS ONE: Evolution in Quantum Leaps: Multiple Combinatorial Transfers of HPI and Other Genetic Modules in Enterobacteriaceae 17 January 2010 19:019 UTC www.plosone.org [Source type: Academic]

    ^ Therefore, only the intB gene sequences were used for genetic comparison.
    • PLoS ONE: Evolution in Quantum Leaps: Multiple Combinatorial Transfers of HPI and Other Genetic Modules in Enterobacteriaceae 17 January 2010 19:019 UTC www.plosone.org [Source type: Academic]

    .Streamlined derivatives of UFW are LaNe RAGE (lariat-dependent nested PCR for rapid amplification of genomic DNA ends),[34] 5'RACE LaNe[35] and 3'RACE LaNe.^ PCR amplification of essentially all sequences increases the amount of DNA for study.
    • PCR, DNA sequencing and in vitro mutagenesis -- Human Molecular Genetics 2 -- NCBI Bookshelf 17 January 2010 19:019 UTC www.ncbi.nlm.nih.gov [Source type: Academic]

    ^ A, Tracheal (Trach) aspirate obtained from patient 25 (Table 2 ); PCR amplification of 375-bp amplimer is seen in EBV-positive ( ) control and tracheal aspirate lanes.
    • Tracheal Aspirate as a Substrate for Polymerase Chain Reaction Detection of Viral Genome in Childhood Pneumonia and Myocarditis -- Akhtar et al. 99 (15): 2011 -- Circulation 16 January 2010 13:52 UTC circ.ahajournals.org [Source type: Academic]

    ^ In the 1980s, he invented the polymerase chain reaction (PCR), a central technique in molecular biology which allows the amplification of specified DNA sequences.

    [36]

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.^ The supernatant was used as DNA template.
  • PLoS Neglected Tropical Diseases: T. cruzi OligoC-TesT: A Simplified and Standardized Polymerase Chain Reaction Format for Diagnosis of Chagas Disease 16 January 2010 13:52 UTC www.plosntds.org [Source type: Academic]

^ A primase, which generates an RNA primer to be used in DNA replication.

^ When multiple primer sets were used, reproducible patterns of amplified complementary DNA fragments were obtained that showed strong dependence on sequence specificity of either primer.
  • Differential display of eukaryotic messenger RNA by means of the polymerase chain reaction -- Liang and Pardee 257 (5072): 967 -- Science 16 January 2010 13:52 UTC www.sciencemag.org [Source type: Academic]

[37] .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.^ PCR was invented by Kary Mullis .
  • Polymerase chain reaction - Hwiki 16 January 2010 13:52 UTC hwiki.fzk.de [Source type: Academic]

^ PCR is an acronym which stands for polymerase chain reaction.

^ More recently the polymerase chain reaction (PCR) was introduced.
  • Journal of Applied Oral Science - Reverse transcription and polymerase chain reaction: principles and applications in dentistry 16 January 2010 13:52 UTC www.scielo.br [Source type: Academic]

[38]
.At the core of the PCR method is the use of a suitable DNA polymerase able to withstand the high temperatures of >90 °C (194 °F) required for separation of the two DNA strands in the DNA double helix after each replication cycle.^ The two double-stranded DNA molecules (DNA ladders) separate into four strands.

^ The double-stranded DNA has to be heated to 94-96C in order to separate the strands.

^ A DNA polymerase is an enzyme that assists in DNA replication.

.The DNA polymerases initially employed for in vitro experiments presaging PCR were unable to withstand these high temperatures.^ High success-rate DNA polymerase .
  • Toyobo Life Science Department -Products PCR- 17 January 2010 19:019 UTC www.toyobo.co.jp [Source type: Academic]

^ High Speed & Efficient DNA polymerase .
  • Toyobo Life Science Department -Products PCR- 17 January 2010 19:019 UTC www.toyobo.co.jp [Source type: Academic]

^ The elongation temperature depends on the DNA-Polymerase.
  • What Is Pcr? 17 January 2010 19:019 UTC www.bionewsonline.com [Source type: Academic]
  • Polymerase chain reaction - Biocrawler, the free encyclopedia 17 January 2010 19:019 UTC www.biocrawler.com [Source type: Academic]

[2] .So the early procedures for DNA replication were very inefficient, time consuming, and required large amounts of DNA polymerase and continual handling throughout the process.^ A DNA polymerase is an enzyme that assists in DNA replication.

^ These remain attached to the DNA throughout the replication process.

^ ELISA-PCR is very time consuming (8 h).
  • Critical Care | Full text | Quantitation of Pseudomonas aeruginosa in wound biopsy samples: from bacterial culture to rapid `real-time' polymerase chain reaction 16 January 2010 13:52 UTC ccforum.com [Source type: Academic]

.The discovery in 1976 of Taq polymerase (a DNA polymerase purified from the thermophilic bacterium, Thermus aquaticus, which naturally occurs in hot (50 to 80 °C (122 to 176 °F)) environments[10]) paved the way for dramatic improvements of the PCR method.^ Chien: Deoxyribonucleic acid polymerase from the extreme thermophile Thermus aquaticus.
  • Journal of Biomedical Discovery and Collaboration | Full text | The effects of business practices, licensing, and intellectual property on development and dissemination of the polymerase chain reaction: case study 16 January 2010 13:52 UTC www.j-biomed-discovery.com [Source type: Academic]

^ One of the first thermostable DNA polymerases was obtained from Thermus aquaticus and was called "Taq."
  • Polymerase chain reaction - Hwiki 16 January 2010 13:52 UTC hwiki.fzk.de [Source type: Academic]

^ DNA polymerase occurs naturally in living organisms.
  • Polymerase chain reaction - Hwiki 16 January 2010 13:52 UTC hwiki.fzk.de [Source type: Academic]

.The DNA polymerase isolated from T. aquaticus is stable at high temperatures remaining active even after DNA denaturation,[11] thus obviating the need to add new DNA polymerase after each cycle.^ For this reason DNA polymerase needs a primer at which it can add the first nucleotide.

^ DNA polymerase can only synthesize new DNA from the 5 to 3 (of the new DNA).

^ The elongation temperature depends on the DNA-Polymerase.

[3] .This allowed an automated thermocycler-based process for DNA amplification.^ Nazarenko IA, Bhatnagar SK, Hohman RJ: A closed tube format for amplification and detection of DNA based on energy transfer.
  • Critical Care | Full text | Quantitation of Pseudomonas aeruginosa in wound biopsy samples: from bacterial culture to rapid `real-time' polymerase chain reaction 16 January 2010 13:52 UTC ccforum.com [Source type: Academic]

^ He became head of the DNA synthesis lab in 1981, and automated the production of oligonucleotides, making the process of DNA synthesis much less laborious.
  • Journal of Biomedical Discovery and Collaboration | Full text | The effects of business practices, licensing, and intellectual property on development and dissemination of the polymerase chain reaction: case study 16 January 2010 13:52 UTC www.j-biomed-discovery.com [Source type: Academic]

^ This shows a typical output of sequence data from an AB1377 automated DNA sequencer as a succession of dye-specific (and therefore base-specific) intensity profiles.
  • PCR, DNA sequencing and in vitro mutagenesis -- Human Molecular Genetics 2 -- NCBI Bookshelf 17 January 2010 19:019 UTC www.ncbi.nlm.nih.gov [Source type: Academic]

.When Mullis developed the PCR in 1983, he was working in Emeryville, California for Cetus Corporation, one of the first biotechnology companies.^ Patent wars The PCR technique was patented by Cetus Corporation, where Mullis worked when he invented the technique.

^ In December 1989, Science selected PCR as the major scientific development of the year and dubbed the Taq enzyme its first annual "Molecule of the Year [ 21 ]."
  • Journal of Biomedical Discovery and Collaboration | Full text | The effects of business practices, licensing, and intellectual property on development and dissemination of the polymerase chain reaction: case study 16 January 2010 13:52 UTC www.j-biomed-discovery.com [Source type: Academic]

^ The commercial nature of the company also granted researchers the flexibility to work on developing PCR, while allowing Cetus to capitalize financially on its lucrative applications.
  • Journal of Biomedical Discovery and Collaboration | Full text | The effects of business practices, licensing, and intellectual property on development and dissemination of the polymerase chain reaction: case study 16 January 2010 13:52 UTC www.j-biomed-discovery.com [Source type: Academic]

.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.^ The DNA will not be damaged at 7°C after just one night.
  • Polymerase chain reaction - Hwiki 16 January 2010 13:52 UTC hwiki.fzk.de [Source type: Academic]

^ Mullis has written that he conceived of PCR while cruising along the Pacific Coast Highway 1 one night in his car {{ .
  • Polymerase chain reaction - Hwiki 16 January 2010 13:52 UTC hwiki.fzk.de [Source type: Academic]

^ There, he was charged with making short chains of DNA for other scientists.
  • Polymerase chain reaction - Hwiki 16 January 2010 13:52 UTC hwiki.fzk.de [Source type: Academic]

[39] .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 DNA polymerase.^ 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.
  • Polymerase chain reaction - Hwiki 16 January 2010 13:52 UTC hwiki.fzk.de [Source type: Academic]

^ Dna test methods - the polymerase chain...
  • Polymerase Chain Reaction - Kosmix : Reference, Videos, Images, News, Shopping and more... 16 January 2010 13:52 UTC www.kosmix.com [Source type: Academic]

^ RNA can be amplified by Taq DNA polymerase.

.In Scientific American, Mullis summarized the procedure: "Beginning with a single molecule of the genetic material DNA, the PCR can generate 100 billion similar molecules in an afternoon.^ As Mullis has written in the Scientific American : "Beginning with a single molecule of the genetic material DNA, the PCR can generate 100 billion similar molecules in an afternoon.
  • Polymerase chain reaction - Hwiki 16 January 2010 13:52 UTC hwiki.fzk.de [Source type: Academic]

^ The beauty of PCR is it is so powerful that one can amplify from a single DNA molecule.
  • UNIT 8: POLYMERASE CHAIN REACTION (PCR) 16 January 2010 13:52 UTC www.med.unc.edu [Source type: Academic]

^ There are three basic steps in PCR. First, the target genetic material must be denatured-that is, the strands of its helix must be unwound and separated-by heating to 90-96°C. The second step is hybridization or annealing, in which the primers bind to their complementary bases on the now single-stranded DNA. The third is DNA synthesis by a polymerase.

.The reaction is easy to execute.^ The reaction is easy to execute.
  • Polymerase chain reaction - Hwiki 16 January 2010 13:52 UTC hwiki.fzk.de [Source type: Academic]

.It requires no more than a test tube, a few simple reagents, and a source of heat."^ It requires no more than a test tube, a few simple reagents, and a source of heat."
  • Polymerase chain reaction - Hwiki 16 January 2010 13:52 UTC hwiki.fzk.de [Source type: Academic]

^ It can, for example, detect the AIDS virus sooner, during the first few weeks after infection, than the standard ELISA test.
  • The polymerase chain reaction -- Powledge 28 (2): 44 -- Advances in Physiology Education 16 January 2010 13:52 UTC advan.physiology.org [Source type: Academic]

^ Nowadays, we have instruments for changing temperature that are much more sophisticated, but it is fundamentally no different than having a few pots of hot water at different temperatures.
  • Bio572: The Polymerase Chain Reaction 16 January 2010 13:52 UTC www.escience.ws [Source type: FILTERED WITH BAYES]

[40] .He was awarded the Nobel Prize in Chemistry in 1993 for his invention,[4] seven years after he and his colleagues at Cetus first put his proposal to practice.^ He was awarded the Nobel Prize in Chemistry in 1993 for his invention, only seven years after he and his colleagues at Cetus first reduced his proposal to practice.
  • Polymerase chain reaction - Hwiki 16 January 2010 13:52 UTC hwiki.fzk.de [Source type: Academic]

^ Nobel prizes are not awarded posthumously.

^ He was awarded the Nobel Prize in Chemistry and the Japan Prize for this work in 1993.

.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 below).^ This discovery is now seen as such an obvious concept that some scientists question if Mullis deserved the Nobel Prize for such a "simple" and "obvious" discovery.
  • CHAPTER #10: GENETIC ENGINEERING 16 January 2010 13:52 UTC www.slic2.wsu.edu:82 [Source type: FILTERED WITH BAYES]

^ The argument could be made that a scientist could better spend his/her time working on cancer or some other terrible disease that afflicts humankind.
  • CHAPTER #10: GENETIC ENGINEERING 16 January 2010 13:52 UTC www.slic2.wsu.edu:82 [Source type: FILTERED WITH BAYES]

^ (See genetic code for further details about codons) Use of degenerate primers can greatly reduce the spesificity of the PCR amplification.

Patent wars

.The PCR technique was patented by Kary Mullis and assigned to Cetus Corporation, where Mullis worked when he invented the technique in 1983. The Taq polymerase enzyme was also covered by patents.^ PCR was invented by Kary Mullis .
  • Polymerase chain reaction - Hwiki 16 January 2010 13:52 UTC hwiki.fzk.de [Source type: Academic]

^ Patent wars The PCR technique was patented by Cetus Corporation, where Mullis worked when he invented the technique.

^ Polymerase chain reaction was invented by Kary Mullis {{ .
  • Polymerase chain reaction - Hwiki 16 January 2010 13:52 UTC hwiki.fzk.de [Source type: Academic]

.There have been several high-profile lawsuits related to the technique, including an unsuccessful lawsuit brought by DuPont.^ There have been several high-profile lawsuits related to the technique, including an unsuccessful lawsuit brought by DuPont .
  • Polymerase chain reaction - Hwiki 16 January 2010 13:52 UTC hwiki.fzk.de [Source type: Academic]

^ There have been several high-profile lawsuits related to the technique, including most famously a lawsuit brought by DuPont.

^ Amplicon LH-PCR is one of several DNA profiling techniques that can be applied to the study of microbial community dynamics and diversity.
  • Assessing Microbial Community Diversity Using Amplicon Length Heterogeneity Polymerase Chain Reaction -- Mills et al. 71 (2): 572 -- Soil Science Society of America Journal 16 January 2010 13:52 UTC soil.scijournals.org [Source type: Academic]

.The pharmaceutical company Hoffmann-La Roche purchased the rights to the patents in 1992 and currently holds those that are still protected.^ The pharmaceutical company Hoffmann-La Roche purchased the rights to the patents in 1992 and currently holds them.

^ The pharmaceutical company Hoffmann-La Roche purchased the rights to the patents in 1992 and currently holds those that are still protected.
  • Polymerase chain reaction - Hwiki 16 January 2010 13:52 UTC hwiki.fzk.de [Source type: Academic]

^ A related patent battle over the Taq polymerase enzyme is still ongoing in several jurisdictions around the world between Roche and Promega .
  • Polymerase chain reaction - Hwiki 16 January 2010 13:52 UTC hwiki.fzk.de [Source type: Academic]

.A related patent battle over the Taq polymerase enzyme is still ongoing in several jurisdictions around the world between Roche and Promega.^ A related patent battle over the Taq polymerase enzyme is still ongoing in several jurisdictions around the world between Roche and Promega .
  • Polymerase chain reaction - Hwiki 16 January 2010 13:52 UTC hwiki.fzk.de [Source type: Academic]

^ The Taq polymerase enzyme is also covered by patents.
  • What Is Pcr? 17 January 2010 19:019 UTC www.bionewsonline.com [Source type: Academic]
  • Polymerase chain reaction - Hwiki 16 January 2010 13:52 UTC hwiki.fzk.de [Source type: Academic]

^ What's the difference between MasterTaq and Taq polymerase?
  • Brinkmann Instruments Canada ::: FAQs-PCR 17 January 2010 19:019 UTC www.brinkmanncanada.com [Source type: FILTERED WITH BAYES]

The legal arguments have extended beyond the lives of the original PCR and Taq polymerase patents, which expired on March 28, 2005[41]

References

  1. ^ Bartlett & Stirling (2003)—A Short History of the Polymerase Chain Reaction. In: Methods Mol Biol. 226:3-6
  2. ^ a b Saiki, RK; Scharf S, Faloona F, Mullis KB, Horn GT, Erlich HA, Arnheim N (December 20 1985). "Enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia". Science 230 (4732): 1350–4. doi:10.1126/science.2999980. PMID 2999980. http://sunsite.berkeley.edu/cgi-bin/ebind2html/pcr/034. 
  3. ^ a b Saiki, RK; Gelfand DH, Stoffel S, Scharf SJ, Higuchi R, Horn GT, Mullis KB, Erlich HA (1988). "Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase". Science 239: 487–91. doi:10.1126/science.2448875. PMID 2448875. http://sunsite.berkeley.edu/cgi-bin/ebind2html/pcr/009. 
  4. ^ a b Kary Mullis Nobel Lecture, December 8, 1993
  5. ^ 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. doi:10.1073/pnas.91.12.5695. PMID 8202550. 
  6. ^ 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
  7. ^ 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. doi:10.1016/j.tibtech.2004.02.011. PMID 15109812. 
  8. ^ Rychlik W, Spencer WJ, Rhoads RE (1990). "Optimization of the annealing temperature for DNA amplification in vitro". Nucl Acids Res 18: 6409–6412. doi:10.1093/nar/18.21.6409. PMID 2243783. http://www.pubmedcentral.nih.gov/articlerender.fcgi?tool=pubmed&pubmedid=2243783. 
  9. ^ 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. doi:10.1038/nbt0594-506. 
  10. ^ a b Chien A, Edgar DB, Trela JM (1976). "Deoxyribonucleic acid polymerase from the extreme thermophile Thermus aquaticus". J. Bacteriol 174: 1550–1557. PMID 8432. 
  11. ^ a b Lawyer FC, Stoffel S, Saiki RK, Chang SY, Landre PA, Abramson RD, Gelfand DH (1993). "High-level expression, purification, and enzymatic characterization of full-length Thermus aquaticus DNA polymerase and a truncated form deficient in 5' to 3' exonuclease activity". PCR Methods Appl. 2: 275–287. PMID 8324500. 
  12. ^ Campbell Biology,7th edition
  13. ^ PCR from problematic templates. Focus 22:1 p.10 (2000).
  14. ^ Helpful tips for PCR. Focus 22:1 p.12 (2000).
  15. ^ 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 0-7637-3383-0. 
  16. ^ "Chemical Synthesis, Sequencing, and Amplification of DNA (class notes on MBB/BIO 343)". Arizona State University. http://photoscience.la.asu.edu/photosyn/courses/BIO_343/lecture/DNAtech.html. Retrieved 2007-10-29. 
  17. ^ Newton CR, Graham A, Heptinstall LE, Powell SJ, Summers C, Kalsheker N, Smith JC, and Markham AF (1989). "Analysis of any point mutation in DNA. The amplification refractory mutation system (ARMS)". Nucleic Acids Research 17 (7): 2503–2516. doi:10.1093/nar/17.7.2503. PMID 2785681. 
  18. ^ Stemmer WP, Crameri A, Ha KD, Brennan TM, Heyneker HL (1995). "Single-step assembly of a gene and entire plasmid from large numbers of oligodeoxyribonucleotides". Gene 164: 49–53. doi:10.1016/0378-1119(95)00511-4. PMID 7590320. 
  19. ^ Innis MA, Myambo KB, Gelfand DH, Brow MA. (1988). "DNA sequencing with Thermus aquaticus DNA polymerase and direct sequencing of polymerase chain reaction-amplified DNA". Proc Natl Acad Sci USA 85: 9436–4940. doi:10.1073/pnas.85.24.9436. PMID 3200828. 
  20. ^ Pierce KE and Wangh LJ (2007). "Linear-after-the-exponential polymerase chain reaction and allied technologies Real-time detection strategies for rapid, reliable diagnosis from single cells". Methods Mol Med. 132: 65–85. doi:10.1007/978-1-59745-298-4_7. PMID 17876077. 
  21. ^ Myriam Vincent, Yan Xu and Huimin Kong (2004). "Helicase-dependent isothermal DNA amplification". EMBO reports 5 (8): 795–800. doi:10.1038/sj.embor.7400200. 
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