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Polymerase Chain Reaction
 

Polymerase Chain Reaction

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

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

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    Polymerase Chain Reaction Polymerase Chain Reaction Presentation Transcript

    • Polymerase Chain Reaction
      • A process used to artificially multiply a chosen piece of genetic material.
      • May also be known as DNA amplification.
      • One strand of DNA may yield 2 30 strands or more.
    • Uses of PCR
      • DNA sequencing.
      • Gene cloning.
      • DNA profiling.
      • Transformation.
      • Making artificial genes.
    • DNA Selection
      • DNA is selected either as a complete chromosome or a fragment.
      • Primers are constructed that will bind within a desired region (purple).
      • Additional reaction chemicals are added.
    • The Reaction Mixture
      • Water and pH buffer
      • DNA to be multiplied
      • RNA Primers
      • Nucleotides
      • DNA Polymerase (Taq)
      A T G C A T C T A C G
    • PCR Machine
      • When mixed, the reaction tubes are placed into a PCR machine.
      • The machine can be set to accurately control reaction times and changes in temperature.
    • Splitting DNA
      • DNA is heated to 95 0 C which causes double stranded DNA to become single stranded.
    • Adding Primers
      • RNA primers are prepared that base pair with a selected sequence of DNA.
      • Two primers must be used.One for each strand of the DNA.
      • The reaction temperature is lowered to 60 0 C to allow the primers to attach ( anneal ).
      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’
    • Adding Nucleotides
      • The reaction temperature is raised to 72 0 C to allow nucleotides to be added.
      • Polymerase enzymes (Taq) catalyse the addition of nucleotides.
      • Nucleotides are added in a 5’ to 3’ direction.
      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’
    • Repeating the cycle
      • In the next cycle the heating and cooling steps are repeated.
      • The original (red/purple) strands reproduce as per the first cycle.
      • The new strands only duplicate between the primer sites to produce blocks of a set length.
      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
    • 1 3 2
    • Continuing the Cycle
      • The cycle is then repeated over and over again.
      • With each cycle the number of short fragments rapidly increases while the number of larger fragments increases slowly.
      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
    • 30 Cycles
      • After 30 cycles, if replication has occurred fully, a total of 2,147,483,648 strands could be produced.
      • All but 62 will be the short length of the desired DNA fragment.
    • 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
    • Questions
      • What does PCR stand for?
      • Polymerase chain reaction.
      • The PCR reaction may also be known as?
      • DNA amplification.
      • In addition to DNA what are the key reactants needed for PCR?
      • pH Buffer, Nucleotides, Taq enzyme, Primers.
      • The first step in the PCR reaction is to heat to 90 0 C. What does this do?
      • Splits double stranded DNA into single strands.
    • Questions Continued
      • The next step is to lower the temperature to 60 0 C. What is the purpose of this?
      • Allows primers to be added.
      • What name is given to the process of joining primers to DNA?
      • Annealing.
      • At what temperature are nucleotides added?
      • 72 0 C
      • What name is given to the polymerase enzyme used?
      • Taq
    • Questions continued
      • In which direction are nucleotides added?
      • 5’ to 3’
      • Why is the cycle repeated many times?
      • To allow the rapid build up of fragment numbers.
      • Name five key uses of PCR.
      • DNA sequencing, gene cloning, DNA profiling transformation, making artificial genes.