polymerase chain reaction

Examples of polymerase chain reaction in the following topics:

• Coupling Specific Genes to Specific Organisms Using PCR

  • Polymerase chain reaction (PCR) is a useful technique for scientists, because it allows for the amplification and mutation of DNA.
  • Describe how polymerase chain reaction (PCR) allows for the amplification and mutation of DNA and enables researchers to study very small samples

• Amplifying DNA: The Polymerase Chain Reaction

  • The polymerase chain reaction (PCR) is a method by which DNA is amplified.
  • The polymerase chain reaction (PCR) is a biochemical technology in molecular biology used to amplify a single, or a few copies, of a piece of DNA across several orders of magnitude, generating thousands to millions of copies of a particular DNA sequence.
  • 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.
  • The reaction produces a limited amount of final amplified product that is governed by the available reagents in the reaction, and the feedback-inhibition of the reaction products.
  • Taq polymerase or another DNA polymerase with a temperature optimum at around 70 °C

• Multiplex and Real-Time PCR

  • Polymerase Chain Reaction (PCR) is a molecular technique commonly used to amplify nucleic acid sequences.
  • The resulting cDNA serves as the template for the PCR reaction.
  • The final product of the reaction is called amplicon.
  • Real-time polymerase chain (RT-PCR) reaction, also called quantitative real-time PCR (qRt-PCR) is used to amplify and quantify targeted DNA molecules.
  • Automated apparatus to amplify DNA sequences using the polymerase chain reaction.

• Classification of Microorganisms

  • The most widely employed methods for classifying microbes are morphological characteristics, differential staining, biochemical testing, DNA fingerprinting or DNA base composition, polymerase chain reaction, and DNA chips.

• Molecular Products from Microbes

  • Taq polymerase is an enzyme that was first isolated from the microbe Thermus aquaticus.
  • The isolation of this polymerase has resulted in the ability to perform polymerase chain reactions (PCR), a process used to amplify DNA segments, in a single step.
  • Prior to the isolation of Taq polymerase, a new DNA polymerase had to be added to the reaction after every cycle because of thermal denaturation.
  • With the addition of Taq polymerase to the reaction tube, the cycle can be performed much more quickly, and less enzyme needs to be used.
  • Describe how Taq polymerase, restriction enzymes and DNA ligase are used in molecular biology

• DNA Analysis Using Genetic Probes and PCR

  • The use of molecular technology in the diagnoses of infectious diseases has been further enhanced by the introduction of gene amplication techniques, such as the polymerase chain reaction (PCR) in which DNA polymerase is able to copy a strand of DNA by elongating complementary strands of DNA that have been initiated from a pair of closely spaced oligonucleotide primers.

• Industrial Microorganisms

  • Specific enzymes which have been isolated and used for industrial purposes include thermostable DNA polymerases from the Pyrococcus furiosus.
  • This type of polymerase isa common tool in molecular biology; it is capable of withstanding the high temperatures that are necessary to complete polymerase chain reactions.

• Synthesizing DNA

  • This occurs in two fashions, by polymerase chain reaction (PCR) which is enzymatic and chemical synthesis.
  • To obtain the desired oligonucleotide, the building blocks are sequentially coupled to the growing oligonucleotide chain in the order required by the sequence of the product.
  • Upon the completion of the chain assembly, the product is released from the solid phase to solution, deprotected, and collected.
  • The occurrence of side reactions sets practical limits for the length of synthetic oligonucleotides (up to about 200 nucleotide residues) because the number of errors accumulates with the length of the oligonucleotide being synthesized.
  • The complex chemical reactions that are needed to couple one nucleotide to another are outlined here.

• Enzymes Used in Industry

  • Enzymes are biological molecules that catalyze (increase the rates of) chemical reactions.
  • Like all catalysts, enzymes work by lowering the activation energy for a reaction, thus dramatically increasing the rate of the reaction.
  • Most enzyme reaction rates are millions of times faster than those of comparable un-catalyzed reactions.
  • As with all catalysts, enzymes are not consumed by the reactions they catalyze, nor do they alter the equilibrium of these reactions.
  • In molecular biology, restriction enzymes, DNA ligase, and polymerases are used to manipulate DNA in genetic engineering, important in pharmacology, agriculture and medicine, and are essential for restriction digestion and the polymerase chain reaction.

• DNA Sequencing Based on Sanger Dideoxynucleotides

  • The classical chain-termination method requires a single-stranded DNA template, a DNA primer, a DNA polymerase, normal deoxynucleotidetriphosphates (dNTPs), and modified nucleotides (dideoxyNTPs) that terminate DNA strand elongation .
  • These chain-terminating nucleotides lack a 3'-OH group required for the formation of a phosphodiester bond between two nucleotides, causing DNA polymerase to cease extension of DNA when a ddNTP is incorporated.
  • The ddNTPs may be radioactively or fluorescently labelled for detection in automated sequencing machines.The DNA sample is divided into four separate sequencing reactions, containing all four of the standard deoxynucleotides (dATP, dGTP, dCTP and dTTP) and the DNA polymerase.
  • Chain-termination methods have greatly simplified DNA sequencing.
  • Dye-terminator sequencing utilizes labelling of the chain terminator ddNTPs, which permits sequencing in a single reaction, rather than four reactions as in the labelled-primer method.