EBio - notes12 - PCR Wiki 12-6

EBio - notes12 - PCR Wiki 12-6 - Polymerase chain reaction...

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Unformatted text preview: Polymerase chain reaction - Wikipedia, the free encyclopedia 12/8/07 3:15 PM Polymerase chain reaction From Wikipedia, the free encyclopedia (Redirected from PCR) The polymerase chain reaction (PCR) is a technique widely used in molecular biology. It derives its name from one of its key components, a DNA polymerase used to amplify (i.e., replicate) a piece of DNA by in vitro enzymatic replication. As PCR progresses, the DNA thus generated is itself used as template for replication. This sets in motion a chain reaction in which the DNA template is exponentially amplified. With PCR it is possible to amplify a single or few copies of a piece of DNA across several orders of magnitude, generating millions or more copies of the DNA piece. PCR can be performed without restrictions on the form of DNA, and it can be extensively modified to perform a wide array of genetic manipulations. A strip of eight PCR tubes, each containing a Almost all PCR applications employ a heat-stable DNA 100l reaction. polymerase, such as Taq polymerase. This DNA polymerase enzymatically assembles a new DNA strand from DNA building blocks, the nucleotides, using single-stranded DNA as template and DNA oligonucleotides (also called DNA primers) required for initiation of DNA synthesis. 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. These different temperature steps are necessary to bring about physical separation of the strands in a DNA double helix (DNA melting), and permit DNA synthesis by the DNA polymerase to selectively amplify the target DNA. The power and selectivity of PCR are primarily due to selecting primers that are highly complementary to the DNA region targeted for amplification, and to the thermal cycling conditions used. 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. 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 forensics and paternity testing); and the detection and diagnosis of infectious diseases. Mullis won the Nobel Prize for his work on PCR. [2] Contents 1 PCR principle and procedure 1.1 Procedure 1.2 PCR optimization http://en.wikipedia.org/wiki/PCR Page 1 of 11 Polymerase chain reaction - Wikipedia, the free encyclopedia 12/8/07 3:15 PM 2 Application of PCR 2.1 Isolation of genomic DNA 2.2 Amplification and quantitation of DNA 3 Variations on the basic PCR technique 4 History 4.1 Patent wars 5 References 6 External links PCR principle and procedure PCR is used to amplify specific regions of a DNA strand (the DNA target). This can be a single gene, a part of a gene, or a non-coding sequence. Most PCR methods typically amplify DNA fragments of up to 10 kilo base pairs (kb), although some techniques allow for amplification of fragments up to 40 kb in size.[3] A basic PCR set up requires several components and reagents. [4] These components include: DNA template that contains the DNA region (target) to be amplified. One or more primers, which are complementary to the DNA regions Figure 1a: An old thermal cycler at the 5' (five prime) and 3' (three prime) ends of the DNA region. for PCR a DNA polymerase such as Taq polymerase or another DNA polymerase with a temperature optimum at around 70C. Deoxynucleotide triphosphates (dNTPs), the building blocks from which the DNA polymerases synthesizes a new DNA strand. Buffer solution, providing a suitable chemical environment for optimum activity and stability of the DNA polymerase. Divalent cations, magnesium or manganese ions; generally Mg 2+ is used, but Mn 2+ can be utilized for PCR-mediated DNA mutagenesis, as higher Mn 2+ concentration increases the error rate during DNA synthesis [5] Monovalent cation potassium ions. The PCR is commonly carried out in a reaction volume of 15-100 l in small reaction tubes (0.2-0.5 ml volumes) in a thermal cycler. The thermal cycler Figure 1b: A very old threeallows heating and cooling of the reaction tubes to control the temperature temperature thermal cycler for PCR required at each reaction step (see below). Thin-walled reaction tubes permit favorable thermal conductivity to allow for rapid thermal equilibration. Most thermal cyclers have heated lids to prevent condensation at the top of the reaction 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. http://en.wikipedia.org/wiki/PCR Page 2 of 11 Polymerase chain reaction - Wikipedia, the free encyclopedia 12/8/07 3:15 PM Procedure The PCR usually consists of a series of 20 to 35 repeated temperature changes called cycles; each cycle typically consists of 2-3 discrete temperature steps. Most commonly PCR is carried out with cycles that have three temperature steps (Fig. 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 temperatures used and the length of time they are applied in each cycle depend on a variety of parameters. 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. [6] Initialization step: This step consists of heating the reaction to a temperature of 94-96C (or 98C if extremely thermostable polymerases are used), which is held for 1-9 minutes. It is only required for DNA polymerases that require heat activation by hot-start PCR. [7] Denaturation step: This step is the first regular cycling event and consists of heating the reaction to 94-98C for 20-30 seconds. It causes melting of DNA template and primers by disrupting the hydrogen bonds between complementary bases of the DNA strands, yielding single strands of DNA. Annealing step: The reaction temperature is lowered to 50-65C for 20-40 seconds allowing annealing of the primers to the single-stranded DNA template. Typically the annealing temperature is about 3-5 degrees Celsius below the Tm of the primers used. Stable DNA-DNA hydrogen bonds are only formed when the primer sequence very closely matches the template sequence. The polymerase binds to the primer-template hybrid and begins DNA synthesis. Figure 2: Schematic drawing of the PCR cycle. (1) Extension/elongation step: The temperature at this Denaturing at 94-96C. (2) Annealing at ~65C step depends on the DNA polymerase used; Taq (3) Elongation at 72C. Four cycles are shown polymerase has its optimum activity temperature at here. 75-80C, [8][9] and commonly a temperature of 72C is used with this enzyme. At this step the DNA polymerase synthesizes a new DNA strand complementary to the DNA template strand by adding http://en.wikipedia.org/wiki/PCR Page 3 of 11 Polymerase chain reaction - Wikipedia, the free encyclopedia 12/8/07 3:15 PM dNTP's 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. The extension time depends both on the DNA polymerase used and on the length of the DNA fragment to be amplified. As a rule-of-thumb, at its optimum temperature, the DNA polymerase will polymerize a thousand bases in one minute. Final elongation: This single step is occasionally performed at a temperature of 70-74C 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 4-15C for an indefinite time may be employed for short-term storage of the reaction. To check whether the PCR generated the anticipated DNA fragment (also sometimes referred to as amplimer), agarose gel electrophoresis is employed for size separation of the PCR products. The size(s) of PCR products is determined by comparison with a DNA ladder, which contains DNA fragments of known size, run on the gel alongside the PCR products (see Fig. 3). PCR optimization In practice, PCR can fail for various reasons, in part due to its sensitivity to contamination causing amplification of spurious DNA products. Because of this, a number of techniques and procedures have been developed for optimizing PCR conditions.[10] [11] Contamination with extraneous DNA is addressed with lab protocols and procedures that separate pre-PCR reactions from potential DNA contaminants. [4] This usually involves spatial separation of PCR-setup areas from areas for analysis or purification of PCR products, and thoroughly cleaning the work surface between reaction setups. Primer-design techniques are important in improving PCR product yield and in avoiding the formation of spurious products, and the usage of alternate buffer components or polymerase enzymes can help with amplification of long or otherwise problematic regions of DNA. Figure 3: Ethidium bromide-stained PCR products after gel electrophoresis. Two sets of primers were used to amplify a target sequence from three different tissue samples. No amplification is present in sample #1; DNA bands in sample #2 and #3 indicate successful amplification of the target sequence. 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. Application of PCR Isolation of genomic DNA 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 Southern and northern blotting and DNA cloning, that require large amounts of DNA, representing a specific DNA region. PCR supplies these techniques with http://en.wikipedia.org/wiki/PCR Page 4 of 11 ...
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