Polymerase Chain Reaction

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simple description of PCR including questions and answers at the end.

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Polymerase Chain Reaction

  1. 1. Polymerase Chain Reaction <ul><li>A process used to artificially multiply a chosen piece of genetic material. </li></ul><ul><li>May also be known as DNA amplification. </li></ul><ul><li>One strand of DNA may yield 2 30 strands or more. </li></ul>
  2. 2. Uses of PCR <ul><li>DNA sequencing. </li></ul><ul><li>Gene cloning. </li></ul><ul><li>DNA profiling. </li></ul><ul><li>Transformation. </li></ul><ul><li>Making artificial genes. </li></ul>
  3. 3. DNA Selection <ul><li>DNA is selected either as a complete chromosome or a fragment. </li></ul><ul><li>Primers are constructed that will bind within a desired region (purple). </li></ul><ul><li>Additional reaction chemicals are added. </li></ul>
  4. 4. The Reaction Mixture <ul><li>Water and pH buffer </li></ul><ul><li>DNA to be multiplied </li></ul><ul><li>RNA Primers </li></ul><ul><li>Nucleotides </li></ul><ul><li>DNA Polymerase (Taq) </li></ul>A T G C A T C T A C G
  5. 5. PCR Machine <ul><li>When mixed, the reaction tubes are placed into a PCR machine. </li></ul><ul><li>The machine can be set to accurately control reaction times and changes in temperature. </li></ul>
  6. 6. Splitting DNA <ul><li>DNA is heated to 95 0 C which causes double stranded DNA to become single stranded. </li></ul>
  7. 7. Adding Primers <ul><li>RNA primers are prepared that base pair with a selected sequence of DNA. </li></ul><ul><li>Two primers must be used.One for each strand of the DNA. </li></ul><ul><li>The reaction temperature is lowered to 60 0 C to allow the primers to attach ( anneal ). </li></ul>G C A U A 5’ 5’ G C A U A 5’ T A G G C A T A G C C T T A T C C G T A T T C G T A T C C G T A T C G G A A T A G G C A T A A G C A 5’
  8. 8. Adding Nucleotides <ul><li>The reaction temperature is raised to 72 0 C to allow nucleotides to be added. </li></ul><ul><li>Polymerase enzymes (Taq) catalyse the addition of nucleotides. </li></ul><ul><li>Nucleotides are added in a 5’ to 3’ direction. </li></ul>A T C C G T A T C G G A A T A G 5’ T A G G C A T A G C C T T A T C C G T A T T C G T G C A U A 5’ G C C T T A T C C G T A T T C G A 5’ G C A U A A T C C G T A T C G G A A T A G G C A T A A G C A 5’
  9. 9. Repeating the cycle <ul><li>In the next cycle the heating and cooling steps are repeated. </li></ul><ul><li>The original (red/purple) strands reproduce as per the first cycle. </li></ul><ul><li>The new strands only duplicate between the primer sites to produce blocks of a set length. </li></ul>A T C C G T A T C G G A A T A G G C A U A 5’ C G T A T C G G A A T A G G C C T T A T C C G T A T T C G A 5’ G C A U A G C A U A 5’ 5’ G C A U A G C C T T A T C C G T A T
  10. 10. 1 3 2
  11. 11. Continuing the Cycle <ul><li>The cycle is then repeated over and over again. </li></ul><ul><li>With each cycle the number of short fragments rapidly increases while the number of larger fragments increases slowly. </li></ul>10 9 8 7 6 5 4 3 2 1 0 Cycle No 2026 1004 494 240 114 52 22 8 2 0 0 Short Fragments 22 20 18 16 14 12 10 8 6 4 2 Long fragments
  12. 12. 30 Cycles <ul><li>After 30 cycles, if replication has occurred fully, a total of 2,147,483,648 strands could be produced. </li></ul><ul><li>All but 62 will be the short length of the desired DNA fragment. </li></ul>
  13. 13. Summary Heat to 95 0 C Denature DNA Cool to 60 0 C Anneal Primers PCR Cycle Add Nucleotides Heat to 72 0 C Heat to 95 0 C Denature DNA Cool to 60 0 C Anneal Primers Heat to 72 0 C Add Nucleotides
  14. 14. Questions <ul><li>What does PCR stand for? </li></ul><ul><li>Polymerase chain reaction. </li></ul><ul><li>The PCR reaction may also be known as? </li></ul><ul><li>DNA amplification. </li></ul><ul><li>In addition to DNA what are the key reactants needed for PCR? </li></ul><ul><li>pH Buffer, Nucleotides, Taq enzyme, Primers. </li></ul><ul><li>The first step in the PCR reaction is to heat to 90 0 C. What does this do? </li></ul><ul><li>Splits double stranded DNA into single strands. </li></ul>
  15. 15. Questions Continued <ul><li>The next step is to lower the temperature to 60 0 C. What is the purpose of this? </li></ul><ul><li>Allows primers to be added. </li></ul><ul><li>What name is given to the process of joining primers to DNA? </li></ul><ul><li>Annealing. </li></ul><ul><li>At what temperature are nucleotides added? </li></ul><ul><li>72 0 C </li></ul><ul><li>What name is given to the polymerase enzyme used? </li></ul><ul><li>Taq </li></ul>
  16. 16. Questions continued <ul><li>In which direction are nucleotides added? </li></ul><ul><li>5’ to 3’ </li></ul><ul><li>Why is the cycle repeated many times? </li></ul><ul><li>To allow the rapid build up of fragment numbers. </li></ul><ul><li>Name five key uses of PCR. </li></ul><ul><li>DNA sequencing, gene cloning, DNA profiling transformation, making artificial genes. </li></ul>

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