Pcr

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Pcr

  1. 1. PCR
  2. 2. ?? <ul><li>A technique in MB, </li></ul><ul><li>A tool for gene amplification </li></ul>
  3. 3. History <ul><li>1985 - Kary Mullis. </li></ul><ul><li>1993 -Nobel Prize in Chemistry. </li></ul><ul><li>Development of a technique/ process by which DNA could be artificially multiplied through repeated cycles of duplication. </li></ul>
  4. 4. Essential components <ul><ul><ul><li>Template ? Needs amplification </li></ul></ul></ul><ul><ul><ul><li>Primers = determine the beginning and end of the region to be amplified </li></ul></ul></ul><ul><ul><ul><li>DNA polymerase </li></ul></ul></ul><ul><ul><ul><li>Buffer = suitable env. </li></ul></ul></ul>
  5. 5. Electrophoresis <ul><li>Components? </li></ul><ul><li>Principle? </li></ul>
  6. 6. PCR Process <ul><li>Template = DNA . </li></ul><ul><li>Enzyme = DNA polymerase. </li></ul><ul><li>Primers = Bind with an end of the desired DNA segment. </li></ul><ul><li>Soln heated - break b onds between strands of DNA. </li></ul><ul><li>Soln cools , the primers bind to the separated strands, and DNA polymerase builds a new strand by joining the free nucleotide bases to the primers. </li></ul><ul><li>Any problem ? </li></ul>
  7. 7. <ul><li>When this process is repeated, a region of DNA between the primers is selectively replicated = New strands formed . </li></ul><ul><li>Further repetitions of the process can produce billions of copies of DNA in several hours. </li></ul>
  8. 8. DNA polymerase <ul><li>occurs naturally in living organisms </li></ul><ul><li>Fx = duplicate DNA during cell division. </li></ul><ul><li>? = binds to a single DNA strand and creating the complementary strand. </li></ul><ul><li>Problem : at high temp DNA polymerase is destroyed, so the enzyme had to be replenished after the heating stage of each cycle </li></ul><ul><li>Inefficient = time consuming, large amounts of DNA polymerase, and continual attention throughout the process. </li></ul>
  9. 9. <ul><li>Discovery of Taq polymerase. </li></ul><ul><li>Hot Springs </li></ul><ul><li>Thermophilic and thermostable. </li></ul><ul><li>Efficient = X add bacteria at each cycle. </li></ul><ul><li>Commercially available by genetically modified bacteria </li></ul>
  10. 10. Primers <ul><li>Short artificial DNA strands </li></ul><ul><li>Nucleotides arranged in a specific order </li></ul><ul><li>Match the beginning and end of the DNA fragment to be amplified </li></ul><ul><li>Fx : They anneal to the DNA template and begins the synthesis of the new DNA strand. </li></ul>
  11. 11. Closer look <ul><li>Denaturing of target material: original template is melted. (90-96 C). Separate strands </li></ul><ul><li>Hybridization : primers bind to their complementary bases to single DNA strands. Primers anneal (45-55C) </li></ul><ul><li>Elongation/ DNA synthesis by RNA polymerase : polymerase makes two new strands ( 72 C), doubling the amount of DNA present. </li></ul>
  12. 12. Cycles <ul><li>Melting phase – High temp </li></ul><ul><li>Annealing phase – Low temp </li></ul><ul><li>Elongation phase –Optimized temp </li></ul>
  13. 13. End product <ul><li>This provides 2 new templates for the next cycle. </li></ul><ul><li>The DNA is again melted, primers anneal, and the Taq makes 4 new strands: </li></ul>
  14. 16. Analysis of product <ul><li>Agarose gel electrophoresis </li></ul><ul><li>NA (-ve) migrate in an electric field. </li></ul><ul><li>Rate of migration is inversely proportional to the size of the nucleic acid fragment. </li></ul><ul><li>Small pieces fast, larger pieces slowly </li></ul>
  15. 17. <ul><li>By comparison with the migration rates of standards of known sizes, it is possible to estimate the size of an uncharacterized fragment of DNA </li></ul>
  16. 18. Advantages <ul><li>Simple – reaction tubes, reagents, heat </li></ul><ul><li>Rapid – Each cycle 1-3 mins. </li></ul><ul><li>Analysis using miniscule amounts. </li></ul><ul><li>Sensitive, accurate, high reliability – Looks directly at DNA rather than searching for clues. </li></ul><ul><li>Unlimited quantities </li></ul><ul><li>Wide range of specimens even old ones. post mortem studies and archaeological studies. </li></ul><ul><li>No need of cloning </li></ul>
  17. 19. Limitations <ul><li>Reaction is sensitive to the levels of divalent cations </li></ul><ul><li>Conditions for each particular application must be worked out = Eg. Wrong annealing time, no binding. Requires optimisation. </li></ul><ul><li>Primer design = must be very specific for the template to be amplified. </li></ul><ul><li>CONTAMINATION = erroneous results. </li></ul><ul><li>Closed environment. </li></ul>
  18. 20. Applications – Human Health <ul><li>Characterize, analyze, synthesize NA. </li></ul><ul><li>Detection of infectious disease organisms – and taxonomic classifications. </li></ul><ul><li>Detection of variations and mutations in genes – Screening of genetic disorders and can provide classification </li></ul>
  19. 21. Faster and reliable diagnostic procedure <ul><li>Can detect bacterial infections even when C &S fails. Faster. </li></ul><ul><li>Certain diagnosis is based on the signs and symptoms . More specific. </li></ul><ul><li>Multiple detection. Eg. can detect 3 different STD on a single swab. </li></ul>
  20. 22. <ul><li>DNA fingerprinting - Layout of DNA fragments. F orensic technique used to identify a person by analysis of DNA from samples eg blood, semen, saliva, hair, etc. </li></ul><ul><li>Paternity testings - Genetic relationships can be determined from two or more genetic fingerprints </li></ul>
  21. 23. Cloning genes <ul><li>The process of isolating a gene from one organism and then inserting it into another organism (GMO). </li></ul><ul><li>PCR amplifies the gene = inserted into a vector </li></ul><ul><li>DNA can then be transferred into A GMO for analysis </li></ul>
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