LAB D.3. Polymerase Chain Reaction- PCR

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LAB D.3. Polymerase Chain Reaction- PCR

• Page 162-175
• Pre Lab Questions are on page 162
• Answer 1,2 3
• (Skip 4)

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This lab is a demonstration

• You will analyze products by gel electrophoresis

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Sequence of Events

• This experiment has considerable Dead Time.
• To finish in reasonable time the TAs will set up
  the PCR reaction before class.
• Very important Read protocol toward the end of
  the power points.
• We will have a lecture on PCR
• Then make agrarose gels to analyze PCR products

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Today's Protocol

• 1. obtain one Easy Start microfuge tube with
  wax coating(contains dNTPs)
• 2. Add 2.5 microliters of plasmid template
• 3. Add 2.0 microliters of upstream primer
• 4. Add 2.0 microliters of downstream primer
• 5. Add 2.0 microliters of TAQ polymerase
• 6. Add 16.5 microliters H2O
• 7. Label tube and place in Thermal-cycler
• Control has everything except primers

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The Polymerase Chain Reaction

• PCR allows one to amplify a specific DNA sequence
  millions of times in just a few hours
• Employs the basic biochemistry of DNA replication
  particularly the concept of the primer
  extension
• Primer is made complementary to a particular DNA
  sequence
• When bound to that sequence DNA polymerase can
  extend the primer synthesizing a new
  complementary DNA strand.

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PCR

• During PCR, high temperature is used to separate
  the DNA molecules into single strands.
• Synthetic single stranded DNA sequences of about
  20 nucleotides complementary to specific DNA
  sequences serve as primers.
• Two primers are used to bracket the target
  sequence to be amplified
• One comp to one strand at the beginning (sense)
• One comp to the other strand at the end (anti
  sense)

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Performing PCR

• Very small amount of DNA sample is mixed with a
  buffer solution containing DNA polymerase and
  primers
• Polymerase (TAQ) is isolated from a thermophylic
  bacteria so it will not be denatured at high
  temperature
• The mixture is taken through replication cycles
  consisting of
• Time at 94C to denature DNA
• Time at 50-60C to allow primers to Anneal
• Time at 72 C during which DNA polymerase can
  extend the primers producing complementary
  strands
• The DNA sequence between the primers doubles
  after each cycle.

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Agarose Gels

• See page 167 of the manual for preparation

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Sindbis Virus

• Made up of three proteins
• Lipid Bilayer (membrane)
• Plus polarity single stranded RNA
• Is infectious
• Virus is a hybrid
• RNA and Protein produced by virus
• Membrane produced by host cell

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Sindbis Structure
E1 E2
C Membrane
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Cloning Sindbis Genome
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Sindbis Membrane Proteins
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Sindbis Genome
Structural Genes
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Amplification

• We will use primers which flank the gene that
  codes for E2

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Sindbis Genome
E2 Gene
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From Monday 2005
Marker Cont Exp
Template DNA Target DNA
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Monday 2006