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.

• Basic Techniques to Manipulate Genetic Material (DNA and RNA)

  • The DNA can be replicated by the DNA polymerase enzyme.
  • Polymerase chain reaction (PCR) is a technique used to amplify specific regions of DNA for further analysis .
  • Polymerase chain reaction, or PCR, is used to amplify a specific sequence of DNA.
  • Primers—short pieces of DNA complementary to each end of the target sequence—are combined with genomic DNA, Taq polymerase, and deoxynucleotides.
  • Taq polymerase is a DNA polymerase isolated from the thermostable bacterium Thermus aquaticus that is able to withstand the high temperatures used in PCR.

• 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

• 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.

• Types and Functions of Proteins

  • Salivary amylase is an enzyme in the mouth that breaks down starch (a long carbohydrate chain) into amylose (a short chain of glucose molecules).
  • These long chains of amino acids are critically important for:
  • Enzymes are proteins that catalyze biochemical reactions, which otherwise would not take place.
  • The substrates are the reactants that undergo the chemical reaction catalyzed by the enzyme.
  • A catabolic enzyme reaction showing the substrate matching the exact shape of the active site.

• DNA Sequencing Techniques

  • The Sanger method is also known as the dideoxy chain termination method.
  • This sequencing method is based on the use of chain terminators, the dideoxynucleotides (ddNTPs).
  • As such the following components are needed: template DNA (which will the be DNA whose sequence will be determined), DNA Polymerase to catalyze the replication reactions, a primer that basepairs prior to the portion of the DNA you want to sequence, dNTPs, and ddNTPs.
  • Most of the time in a Sanger sequencing reaction, DNA Polymerase will add a proper dNTP to the growing strand it is synthesizing in vitro.
  • Each sequencing reaction is a modified replication reaction involving flourescently-tagged nucleotides, but no chain-terminating dideoxy nucleotides are needed.

• Elongation and Termination in Prokaryotes

  • The transcription elongation phase begins with the release of the σ subunit from the polymerase.
  • Rho-dependent termination is controlled by the rho protein, which tracks along behind the polymerase on the growing mRNA chain.
  • Near the end of the gene, the polymerase encounters a run of G nucleotides on the DNA template and it stalls.
  • As a result, the rho protein collides with the polymerase.
  • As the polymerase nears the end of the gene being transcribed, it encounters a region rich in C–G nucleotides.