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Polymerase Chain Reaction


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Polymerase Chain Reaction

  1. 1. The Polymerase Chain Reaction (PCR) <ul><li>Polymerase chain reaction ( PCR ) is a revolutionary molecular biology technique for enzymatically replicating DNA </li></ul><ul><li>The technique allows a small amount of the DNA molecule to be amplified many times in an exponential manner </li></ul><ul><li>PCR is commonly used in medical and biological research labs for a variety of tasks, such as the detection of hereditary diseases, the identification of genetic fingerprints, the diagnosis of infectious diseases, the cloning of genes, and paternity testing. </li></ul><ul><li>[Insert diagram] </li></ul>
  2. 3. <ul><li>PCR product compared with DNA ladder in agarose gel </li></ul><ul><li>DNA ladder ( lane 1 ), </li></ul><ul><li>the PCR product in low concentration ( lane 2 ), </li></ul><ul><li>and high concentration ( lane 3 ). </li></ul>
  3. 4. Stages in PCR <ul><li>PCR, as currently practiced, requires several basic components. These components are: </li></ul><ul><li>DNA template, which contains the region of the DNA fragment to be amplified </li></ul><ul><li>Two primers , which determine the beginning and end of the region to be amplified ( primers = short lengths of a known DNA sequence ) </li></ul><ul><li>DNA-Polymerase , which copies the region to be amplified </li></ul><ul><li>Nucleotides , from which the DNA-Polymerase builds the new DNA </li></ul><ul><li>Buffer, which provides a suitable chemical environment for the DNA-Polymerase </li></ul><ul><li>The PCR reaction is carried out in a thermal cycler . This is a machine that heats and cools the reaction tubes within it to the precise temperature required for each step of the reaction. </li></ul>
  4. 5. <ul><li>The PCR process consists of a series of twenty to thirty cycles. Each cycle consists of three steps: </li></ul><ul><li>(1) The double-stranded DNA has to be heated to 94-96°C in order to separate the strands. This step is called denaturing; it breaks apart the hydrogen bonds that connect the two DNA strands. </li></ul><ul><li>(2) After separating the DNA strands, the temperature is lowered so the primers can attach themselves to the single DNA strands. This step is called annealing. The temperature of this stage depends on the primers and is usually 5°C below their melting temperature (45-60°C) </li></ul><ul><li>(3) Finally, the DNA-Polymerase has to fill in the missing strands. It starts at the annealed primer and works its way along the DNA strand. This step is called extension. The extension temperature depends on the DNA-Polymerase </li></ul>Taq DNA Polymerase: This is a thermal stable enzyme isolated from thermophilic bacteria. This enzyme canonly synthesis DNA in one direction 3’ - 5’
  5. 6. Applications of PCR <ul><li>1 ) MOLECULAR BIOLOGICAL RESEARCH - gene screening analysis (looking for a gene) and DNA cloning (copying particular DNA sequences) </li></ul><ul><li>2) GENETIC MAPPING STUDIES e.g. human genome project, sequence tagging on genome sites </li></ul><ul><li>3) CLINICAL & DIAGNOSTIC USES - screening and diagnosis of HIV; cancer (detects mutations of oncogenes); genetic disorders e.g. cystic fibrosis </li></ul><ul><li>4) GENETIC IDENTIFICATION and DNA TYPING - forensic and parentage testing; sex determination of pre-natal cells; classification of species </li></ul><ul><li>5) IDENTIFICATION of TRACE AMOUNTS of DNA - detection of contamination of foodstuff by: food-borne pathogens, genetically modified organisms in food products, presence of pork in beef etc </li></ul>