Polymerase Chain Reaction

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  • 1. Polymerase Chain Reaction “Xeroxing” DNA
  • 2. Life is not fair!
    • 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
    • 1985—the polymerase chain reaction was introduced to the scientific community at a conference in October
  • 3. Still not fair!
    • Cetus rewarded Kary Mullis with a $10,000 bonus for his invention
    • Later, during a corporate reorganization, Cetus sold the patent for the PCR process to a pharmaceutical company Hoffmann-LaRoche for $300 million
    • Again I say, life is not fair!
  • 4. Polymerase Chain Reaction
    • PCR for short!
    • A technique for making MANY copies of a particular DNA sequence
    • Allows us to start with VERY SMALL samples
    • We need a large sample to perform electrophoresis and other analyses
  • 5. PCR: Amplification of DNA
  • 6. PCR: Amplification of DNA
    • Often, only a small amount of DNA is available
      • A drop of blood
      • A rare cell type
    • Two methods currently exist for amplifying the DNA or making copies
      • Cloning—takes a long time for enough clones to reach maturity
      • PCR—works on even a single molecule quickly
  • 7. PCR basis: Directionality of DNA backbone
  • 8. Recall that DNA is antiparallel
  • 9. Heat causes denaturing—H-bonds between base pairs “break”
  • 10. Annealing
    • Fancy word for renaturing
      • When denatured strands of DNA cool, it can renature
      • HYDROGEN BONDS RE-FORM!
      • Complimentary base pairs must line up in order for this to happen
    • The denaturing and annealing of DNA is an important part of PCR
  • 11. DNA Polymerase moves in a 5’  3’ direction
    • DNA polymerase is the key to “xeroxing” copies of the DNA sample!
    • DNA polymerase is an enzyme that uses an existing SINGLE strand of DNA as a template to form a new complementary strand.
    • To get started, DNA polymerase needs a small complementary sequence, called a primer
  • 12. Note: SYNTHESIS occurs in the 5’  3’ direction
  • 13. Where do we get loads of this DNA polymerase enzyme?
    • Most commonly, our good buddy E. coli
    • Trouble is, E. coli ’s DNA polymerase is ALSO denatured at the high temperatures needed to denature the DNA!
    • This problem was solved by looking to bacteria that live in hot springs such as Old Faithful
    • The DNA polymerase of these bacterial cells can withstand the high temperatures needed WITHOUT having the enzyme denature!
  • 14. Old Faithful!
  • 15. Taq polymerase
    • This is the name of the most common heat-resistant polymerase extracted from these thermophilic bacteria.
    • Thermus aquaticus is the genus and species name of the bacteria, Taq for short!
  • 16. Making PCR twice as fast!
    • The stretch of DNA to be amplified is often referred to as the “target sequence”
    • The template DNA is usually a larger stretch of sample DNA that contains the target sequence
    • The primers are short stretches of DNA that flank the target sequence and are complementary to opposite strands of the longer sample DNA
    • 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
    • Big deal?
    • You bet! Now both strands of the sample DNA are templates for the polymerase enzymes!!
  • 17.  
  • 18. PCR: Essential Reagents
    • An excess of spare nucleotides
    • An excess of primers
      • We want the primers to bind to the sample DNA once it is denatured to prevent the strands from reannealing
    • PLENTY of DNA polymerase [Taq polymerase in particular]
    • Sample template of DNA
  • 19.  
  • 20. PCR: Pipetting the reagents into the reaction tube
  • 21. “ PCR machine”: The thermocycler
    • A programmable machine that can change temperatures with great accuracy and at precise times—it’s like a fancy incubator
    • 94 °C is always the first temperature set in the program
      • DNA denatures at this temperature
  • 22. First denaturation
    • As the temperature is raised to 94 °C, the target DNA denatures
    • At these high temperatures, there will be NO BINDING of any sequences
    • The reaction mixture is left at 94°C for 5 minutes to allow the DNA to completely denature
  • 23.  
  • 24. Binding of primers during annealing
    • As the tube is cooled, the primers will bind to the sequences that flank the target sequence on the two strands
  • 25. Thermocycler lowered to annealing temperature
    • The tube is lowered to 50 °C for 2 minutes to allow the primers to anneal to the template DNA.
  • 26. PCR: Polymerization by High –temperature DNA polymerase
    • After the primers have bound to the flanking sequences, the DNA polymerase will start to synthesize the complementary strand.
    • The end result will be four copies of the target sequence, doubling the two present at the start.
  • 27.  
  • 28. Thermocycler at polymerization temperature
    • 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
  • 29. PCR denaturation step repeated
    • When the temperature is again raised to 94 °C, both the original template DNA and the copies from the last cycle denature
  • 30. PCR: Annealing and polymerization repeated
    • Now, both the two original target sequences AND the two copies of the sequence can act as templates.
    • As the reaction is cooled, primers bind to the templates, and the polymerase makes copies in the opposite directions.
    • You can see in this next image why primers are needed in both directions.
  • 31.  
  • 32. Denaturation repeated a third time
    • The temperature is raised yet again, and the double strands fall apart
    • There are now EIGHT template strands available for making copies
  • 33.  
  • 34. Our hero! The thermocycler!
    • The developers of PCR and the early researchers who used the technique spent long hours transferring tubes between the different hot water baths.
    • 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
    • The machine can be programmed for any changes in temperature the experimenter finds useful.
  • 35. Repeat, repeat, repeat!
    • After this denaturing and synthesizing process is repeated for 30 cycles, the sequence will have been amplified a BILLION times
    • 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!
  • 36. After 30 cycles this becomes one billion! 2 30
  • 37. What next?
    • Now that the most minute sample of DNA has been amplified, there is a large enough PCR product to be electrophoresed.
  • 38. Applications of PCR
    • The first application dealt with detection of genetic mutations
    • Now the smallest trace evidence from crime scenes can be amplified in order to provide DNA fingerprinting data