polymerase chain reaction(redirected from Annealing temperature)
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pol·ym·er·ase chain re·ac·tion (PCR),
The replication of DNA in the living cell is facilitated by polymerases. The two DNA chains of the double helix first unzip from one another, and DNA polymerase then generates a copy of each strand by adding free nucleotides to form a sequence of base pairs complementary with the sequence in the strand. The laboratory technique known as PCR, for which the American biochemist Kary Mullis won a Nobel Prize in Chemistry in 1993, exploits the capacity of DNA polymerase to assemble new DNA. Taq polymerase, named for its source, Thermus aquaticus, a thermophilic bacterium, is added to a mixture of free nucleotides and primers. (Primers are specially prepared units containing both RNA and DNA with a free terminus where the polymerase will react.) The short sequence of DNA to be amplified is flanked by two primers. After the reaction begins, the polymerase generates numerous copies of the target sequence. The sequential phases of the reaction are initiated simply by making a series of strategic changes in the temperature of the system. Millions of copies of the target sequence can be generated by cyclically repeating these temperature changes as many as 30 times, each DNA strand produced by 1 cycle giving rise to many more in the next. Coupled with appropriate detection methods, the technique can confirm the presence of any gene or short DNA sequence, even when only a few copies of the target molecule are present in the specimen. PCR technology is both highly sensitive and highly specific, and does not require prior purification of the specimen. It is used in the diagnosis of infectious diseases through identification of microbial pathogens in clinical material, including organisms that are difficult or impossible to culture either by their nature or because of prior treatment with antimicrobial agents. PCR also has application to genetic testing, including surveillance for heritable defects in parents or fetus; to oncology, for detection of mutations in tumor suppressor genes in neoplasms and, in particular, in microscopically benign tumor margins; and to forensic medicine, for DNA fingerprinting and determination of paternity.
polymerase chain reaction
polymerase chain reactionMolecular biology A molecular technique that uses DNA polymerases from high-temperature bacteria–known as extremophiles to rapidly amplify–ie, ↑ the number of copies of–a sequence of DNA in a sample; starting from minimal amounts–<< 1 µg–as little as one copy of a sequence of DNA, PCR exponentially amplifies a target DNA sequence, which has been inserted between 2 oligonucleotide primers through multiple amplification cycles Application Prenatal Dx of hereditary disease–sickle cell anemia, PKU, cystic fibrosis; ID gene rearrangements in lymphoproliferative disorders, determine fetal sex, Lyme disease, TB, Chlamydia trachomatis, ID viruses–HIV, CMV, HPV, HBV, delineate viral link to cancer–HTLV-1, HPV, bacteria, parasites, pathogenic mechanisms–DM, pemphigus vulgaris, myasthenia gravis, oncogene-induced cancer Sensitivity In detecting leukemia in BM–Bx has a 65-75% sensitivity, Southern blot analysis of gene rearrangement, 98-99% sensitivity, PCR, 99.999%. See Allele-specific PCR, AP-PCR, DNA amplification, Fluorophore-enhanced repetitive sequence-based-PCR, Inverse PCR, Jumping PCR, Multiplex PCR, Nested PCR, Reverse-transcriptase PCR, Semi-nested PCR, Touchdown PCR. Cf Ligase chain reaction.
pol·ym·er·ase chain re·ac·tion(PCR) (pŏ-lim'ĕr-ās chān rē-ăk'shŭn)
polymerase chain reactionAn important technique for rapidly producing large numbers of copies of any required sequence of DNA. DNA is separated by heat into its two strands, small molecules called primers are attached to the sequences at either end of the target sequence, and an enzyme, DNA polymerase, is used to build a new strand of the section between the primers. This becomes a template for the production of further strands and in twenty cycles a million copies are made. The polymerase chain reaction is one of the most powerful techniques currently in use in biological science. The American biochemist inventor of the process, Karry B. Mullis, was awarded the Nobel Prize for Chemistry in 1993.
polymerase chain reaction (PCR)a method for amplifying specific DNA sequences in vitro. The technique exploits some of the features of DNA REPLICATION and permits millions or billions of copies of a DNA sequence to be produced within few hours. In order to carry out the basic PCR reaction (see Fig. 256 ), two PRIMERS that flank the DNA region to be amplified (the target) and that hybridize (see MOLECULAR HYBRIDIZATION) to the opposite strands of the double-stranded DNA, are needed. The double-stranded target DNA is heat-denatured (see DENATURATION).to separate the two strands and, on cooling, a primer is annealed to the complementary sequence in each of the strands. The primers are extended using DNA POLYMERASE, which copies each of the TEMPLATE strands. (A heat-stable DNA polymerase, which will not be impaired by heat during later steps in the reaction, and which therefore allows the process to be automated without the need to add enzyme at each cycle, is preferred.
For this reason, a thermo-stable enzyme, such as Taq polymerase obtained from a THERMOPHILIC BACTERIUM, is commonly used.) This produces two double-stranded DNA molecules, which, following their heat-denaturation, can serve as templates for another cycle of amplification. Primers are again annealed and extended by DNA synthesis. In this way, a doubling of the amount of DNA previously present occurs. The termini of the amplified sequence are defined by the primers. A series of cycles involving heat-denaturation, primer annealing and DNA synthesis by primer extension results in the amplification of the target DNA at approximately 2n, where n is the number of cycles. Thus the ‘chain reaction’, once set up, results in the exponential amplification of the starting DNA. Taq polymerase does not have an EDITING function and so errors (in the form of incorrect BASES) may be introduced during DNA synthesis. To reduce the frequency of such errors, the PCR may be started with a large number of DNA template molecules, if available, so that fewer cycles are needed to amplify the DNA. Alternatively, other enzymes, with increased fidelity (which are gradually becoming available) may be used. The PCR is useful for amplifying DNA initially present in very low amounts, so that sufficient is available for subsequent analysis. The applications of the PCR are potentially enormous. It has been used to amplify DNA in, for example, DNA FINGERPRINTING; the analysis of ancient DNA from fossils; mapping of the human GENOME; the detection of microorganisms present in low numbers in food and water samples; and so on.