Polymerase Chain Reaction


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Polymerase Chain Reaction

  1. 1. Polymerase Chain Reaction “Xeroxing” DNA
  2. 2. Life is not fair! <ul><li>1983—Kary Mullis, a scientist working for the Cetus Corporation was driving along US Route 101 in northern California when he came up with the idea for the polymerase chain reaction </li></ul><ul><li>1985—the polymerase chain reaction was introduced to the scientific community at a conference in October </li></ul>
  3. 3. Still not fair! <ul><li>Cetus rewarded Kary Mullis with a $10,000 bonus for his invention </li></ul><ul><li>Later, during a corporate reorganization, Cetus sold the patent for the PCR process to a pharmaceutical company Hoffmann-LaRoche for $300 million </li></ul><ul><li>Again I say, life is not fair! </li></ul>
  4. 4. Polymerase Chain Reaction <ul><li>PCR for short! </li></ul><ul><li>A technique for making MANY copies of a particular DNA sequence </li></ul><ul><li>Allows us to start with VERY SMALL samples </li></ul><ul><li>We need a large sample to perform electrophoresis and other analyses </li></ul>
  5. 5. PCR: Amplification of DNA
  6. 6. PCR: Amplification of DNA <ul><li>Often, only a small amount of DNA is available </li></ul><ul><ul><li>A drop of blood </li></ul></ul><ul><ul><li>A rare cell type </li></ul></ul><ul><li>Two methods currently exist for amplifying the DNA or making copies </li></ul><ul><ul><li>Cloning—takes a long time for enough clones to reach maturity </li></ul></ul><ul><ul><li>PCR—works on even a single molecule quickly </li></ul></ul>
  7. 7. PCR basis: Directionality of DNA backbone
  8. 8. Recall that DNA is antiparallel
  9. 9. Heat causes denaturing—H-bonds between base pairs “break”
  10. 10. Annealing <ul><li>Fancy word for renaturing </li></ul><ul><ul><li>When denatured strands of DNA cool, it can renature </li></ul></ul><ul><ul><li>HYDROGEN BONDS RE-FORM! </li></ul></ul><ul><ul><li>Complimentary base pairs must line up in order for this to happen </li></ul></ul><ul><li>The denaturing and annealing of DNA is an important part of PCR </li></ul>
  11. 11. DNA Polymerase moves in a 5’  3’ direction <ul><li>DNA polymerase is the key to “xeroxing” copies of the DNA sample! </li></ul><ul><li>DNA polymerase is an enzyme that uses an existing SINGLE strand of DNA as a template to form a new complementary strand. </li></ul><ul><li>To get started, DNA polymerase needs a small complementary sequence, called a primer </li></ul>
  12. 12. Note: SYNTHESIS occurs in the 5’  3’ direction
  13. 13. Where do we get loads of this DNA polymerase enzyme? <ul><li>Most commonly, our good buddy E. coli </li></ul><ul><li>Trouble is, E. coli ’s DNA polymerase is ALSO denatured at the high temperatures needed to denature the DNA! </li></ul><ul><li>This problem was solved by looking to bacteria that live in hot springs such as Old Faithful </li></ul><ul><li>The DNA polymerase of these bacterial cells can withstand the high temperatures needed WITHOUT having the enzyme denature! </li></ul>
  14. 14. Old Faithful!
  15. 15. Taq polymerase <ul><li>This is the name of the most common heat-resistant polymerase extracted from these thermophilic bacteria. </li></ul><ul><li>Thermus aquaticus is the genus and species name of the bacteria, Taq for short! </li></ul>
  16. 16. Making PCR twice as fast! <ul><li>The stretch of DNA to be amplified is often referred to as the “target sequence” </li></ul><ul><li>The template DNA is usually a larger stretch of sample DNA that contains the target sequence </li></ul><ul><li>The primers are short stretches of DNA that flank the target sequence and are complementary to opposite strands of the longer sample DNA </li></ul><ul><li>The primer attaches to the beginning of the target sequence on one strand and the END of the target sequence on the other strand of DNA </li></ul><ul><li>Big deal? </li></ul><ul><li>You bet! Now both strands of the sample DNA are templates for the polymerase enzymes!! </li></ul>
  17. 18. PCR: Essential Reagents <ul><li>An excess of spare nucleotides </li></ul><ul><li>An excess of primers </li></ul><ul><ul><li>We want the primers to bind to the sample DNA once it is denatured to prevent the strands from reannealing </li></ul></ul><ul><li>PLENTY of DNA polymerase [Taq polymerase in particular] </li></ul><ul><li>Sample template of DNA </li></ul>
  18. 20. PCR: Pipetting the reagents into the reaction tube
  19. 21. “ PCR machine”: The thermocycler <ul><li>A programmable machine that can change temperatures with great accuracy and at precise times—it’s like a fancy incubator </li></ul><ul><li>94 °C is always the first temperature set in the program </li></ul><ul><ul><li>DNA denatures at this temperature </li></ul></ul>
  20. 22. First denaturation <ul><li>As the temperature is raised to 94 °C, the target DNA denatures </li></ul><ul><li>At these high temperatures, there will be NO BINDING of any sequences </li></ul><ul><li>The reaction mixture is left at 94°C for 5 minutes to allow the DNA to completely denature </li></ul>
  21. 24. Binding of primers during annealing <ul><li>As the tube is cooled, the primers will bind to the sequences that flank the target sequence on the two strands </li></ul>
  22. 25. Thermocycler lowered to annealing temperature <ul><li>The tube is lowered to 50 °C for 2 minutes to allow the primers to anneal to the template DNA. </li></ul>
  23. 26. PCR: Polymerization by High –temperature DNA polymerase <ul><li>After the primers have bound to the flanking sequences, the DNA polymerase will start to synthesize the complementary strand. </li></ul><ul><li>The end result will be four copies of the target sequence, doubling the two present at the start. </li></ul>
  24. 28. Thermocycler at polymerization temperature <ul><li>The temperature of the reaction mixture is raised to 72 °C for 3 minutes to allow the polymerase to copy the target sequence. The temperature is raised to 94°C and the cycle is repeated </li></ul>
  25. 29. PCR denaturation step repeated <ul><li>When the temperature is again raised to 94 °C, both the original template DNA and the copies from the last cycle denature </li></ul>
  26. 30. PCR: Annealing and polymerization repeated <ul><li>Now, both the two original target sequences AND the two copies of the sequence can act as templates. </li></ul><ul><li>As the reaction is cooled, primers bind to the templates, and the polymerase makes copies in the opposite directions. </li></ul><ul><li>You can see in this next image why primers are needed in both directions. </li></ul>
  27. 32. Denaturation repeated a third time <ul><li>The temperature is raised yet again, and the double strands fall apart </li></ul><ul><li>There are now EIGHT template strands available for making copies </li></ul>
  28. 34. Our hero! The thermocycler! <ul><li>The developers of PCR and the early researchers who used the technique spent long hours transferring tubes between the different hot water baths. </li></ul><ul><li>Fortunately, this tedious process is almost entirely automated by the thermocycler. The times and temperatures described in this tutorial for each step are averages, and different experiments call for different conditions </li></ul><ul><li>The machine can be programmed for any changes in temperature the experimenter finds useful. </li></ul>
  29. 35. Repeat, repeat, repeat! <ul><li>After this denaturing and synthesizing process is repeated for 30 cycles, the sequence will have been amplified a BILLION times </li></ul><ul><li>This geometric increase in the amount of target sequence can be achieved in just a few hours rather than the much longer times required by older methods! </li></ul>
  30. 36. After 30 cycles this becomes one billion! 2 30
  31. 37. What next? <ul><li>Now that the most minute sample of DNA has been amplified, there is a large enough PCR product to be electrophoresed. </li></ul>
  32. 38. Applications of PCR <ul><li>The first application dealt with detection of genetic mutations </li></ul><ul><li>Now the smallest trace evidence from crime scenes can be amplified in order to provide DNA fingerprinting data </li></ul>