polymerase chain reaction

Examples of polymerase chain reaction in the following topics:

• 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.

• 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.

• Initiation of Transcription in Eukaryotes

  • Instead of a single polymerase comprising five subunits, the eukaryotes have three polymerases that are each made up of 10 subunits or more.
  • RNA polymerase III is also located in the nucleus.
  • The tRNAs have a critical role in translation: they serve as the adaptor molecules between the mRNA template and the growing polypeptide chain.
  • Not all miRNAs are transcribed by RNA Polymerase II, RNA Polymerase III transcribes some of them.
  • A generalized promoter of a gene transcribed by RNA polymerase II is shown.

• DNA Replication in Prokaryotes

  • One of the key players is the enzyme DNA polymerase, which adds nucleotides one by one to the growing DNA chain that are complementary to the template strand.
  • When the bond between the phosphates is broken, the energy released is used to form the phosphodiester bond between the incoming nucleotide and the growing chain.
  • In prokaryotes, three main types of polymerases are known: DNA pol I, DNA pol II, and DNA pol III.
  • DNA polymerase III uses this primer to synthesize the daughter DNA strand.
  • DNA polymerase I replaces the RNA primer with DNA.

• The Protein Synthesis Machinery

  • The peptidyl-tRNA carrying the growing polypeptide chain is held in the P site.
  • The tRNA molecules are transcribed by RNA polymerase III.
  • In eukaryotes, tRNA mole are transcribed from tRNA genes by RNA polymerase III.
  • The process of pre-tRNA synthesis by RNA polymerase III only creates the RNA portion of the adaptor molecule.
  • These enzymes first bind and hydrolyze ATP to catalyze the formation of a covalent bond between an amino acid and adenosine monophosphate (AMP); a pyrophosphate molecule is expelled in this reaction.

• DNA Replication in Eukaryotes

  • The template strand specifies which of the four DNA nucleotides (A, T, C, or G) is added at each position along the new chain.
  • This process will continue until the DNA polymerase reaches the end of the template strand.
  • DNA polymerase can only synthesize new strands in the 5' to 3' direction.
  • DNA polymerase halts when it reaches a section of DNA template that has already been replicated.
  • An RNA primer is synthesized by primase and is elongated by the DNA polymerase.

• DNA Repair

  • Most mistakes during replication are corrected by DNA polymerase during replication or by post-replication repair mechanisms.
  • DNA replication is a highly accurate process, but mistakes can occasionally occur as when a DNA polymerase inserts a wrong base.
  • The polymerase checks whether the newly-added base has paired correctly with the base in the template strand.
  • Thus, DNA polymerase is able to remove the incorrectly-incorporated bases from the newly-synthesized, non-methylated strand.
  • Spontaneous mutations occur without any exposure to any environmental agent; they are a result of natural reactions taking place within the body.

• The Two Parts of Photosynthesis

  • Light-dependent and light-independent reactions are two successive reactions that occur during photosynthesis.
  • Just as the name implies, light-dependent reactions require sunlight.
  • Photosystems consist of a light-harvesting complex and a reaction center.
  • In photosystem I, the electron comes from the chloroplast electron transport chain.
  • Photosynthesis takes place in two stages: light-dependent reactions and the Calvin cycle (light-independent reactions).