Mandatory Articles/Finding structure on the web.

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Lecture 2

• Mandatory Articles/Finding structure on the web.
• Restriction enzymes
• a) History
• b) uses/properties
• PCR
• a) basics
• b) considerations
• Agarose Gel electrophoresis
• Tips for this weeks lab

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Mandatory Articles

• Sept. 29 Article 1
• Oct. 6 Article 2
• Oct. 13 Thanksgiving
• Oct. 20 Article 3
• Oct. 27 Article 4
• Nov. 3 Article 5
• Nov. 10 Article 6
• Nov. 17 Article 7

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Finding structures on the Web

• http//www.ncbi.nlm.nih.gov/
• Choose structures
• Search for the one you are interested in.
• Download file and viewer and look at your
  structure.

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Socratic Method

• Questions are the fundamental guiding element
  that lead to answers, or boundaries to the
  unanswerable.
• Method of critical thinking/investigation.
• Development of student intellectual autonomy.
• I use a loose Socratic style in lecture.

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Restriction enzymes

• Enzymes and recombinant DNA technology.
• How were restriction enzymes found originally?
• What are the properties of restriction enzymes?
• What are their uses?

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Early 1970s Genomic Studies How to cut DNA into
manageable fragments? Chemical/mechanical means
non-specific, non-reproducible
Breakthrough needed
Restriction Endonucleases Molecular Scissors for
Cutting DNA
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But where did restriction enzymes come from?

• They were discovered as a method of protecting
  bacteria from bacteriophage infection.
• Called the restriction modification system

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Restriction Enzymes Recognize Palindromic
Sequences
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Restriction enzymes

• Considerations
• Why do we have MgCl2 ?
• Why do we have NaCl?
• Why do we have a buffer?

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Restriction enzymes can be used to create a MAP
of DNA

• Cleavage of DNA with restriction enzymes provides
  landmarks and sequence information.

? MAP ?
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Restriction enzymes can be used to create a MAP
of DNA
Restriction enzymes played a pivotal role in
cloning of the Human Genome
Digestion of DNA with restriction enzymes
cloning into vectors
Construction of restriction maps of individual
clones
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FORMATION OF RECOMBINANT DNA
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Polymerase Chain Reaction

• Considerations
• Why do we have MgCl2?
• Why do we have a buffer?
• Why do we have dNTPs?
• Why do we have primers?
• Why three temperatures per cycle?

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POLYMERASE CHAIN REACTION
STEPS INVOLVED
Reverse Primer
Forward Primer
Taq Polymerase
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Unlike most enzymes, Taq DNA polymerase can
withstand high temperatures necessary for DNA
strand separation and can be left in the reaction.
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PCR Representative Temperature Profile
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POLYMERASE CHAIN REACTION
AMPLIFICATION OF DNA
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POLYMERASE CHAIN REACTION (Cont)
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EXPONENTIAL AMPLIFICATION OF PRODUCT
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DESIGNING PRIMERS
1.  Primers should be 18-25 bases in length  
2.  Base composition should be 50-60 (GC)  
3.  Primers should end (3') in CG or GC this
creates tight ends and increases
efficiency of priming   4.  Tm 4(G C) 2(A
T) oC Ta should be 2-5 oC below Tm( 55-60oC)
5.  3'-ends of primers should not be
complementary primer-dimer 6.  Primer
self-complementary (ability to form hairpins)
should be avoided.  7.  Three or more Cs or Gs
at the 3'-ends of may promote mispriming at G or
C-rich sequences (because of stability of
annealing), and should be avoided. 
Adapted from Innis and Gelfand, 1991
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Gel electrophoresis

• Considerations
• On what basis are molecules separated?
• How does a gel work?
• Which way do molecules go and why?
• What colour are electrodes and anodes?

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AGAROSE GEL ELECTROPHORESIS
Agarose A polysaccharide extracted from
seaweed. Used to separate DNA fragments
based on size
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Detecting DNA using Ethidium Bromide
Intercalates between bases of DNA
When excited by UV light, EtBr emits fluorescent
light at 590 nm EtBr is a very dangerous mutagen.
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How are different sizes of DNA strands separated
on agarose gel?
Mixture of DNA molecules
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DNA separation is based on fragment size
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Size markers and their use to determine size
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Size markers and their use to determine size
Mobility of a DNA fragment is proportional to
the log of its size in bases.
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Plot of relative mobility of DNA vs log of size
on semi-log graph paper
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Varying concentrations of agarose
How does one make a 0.7, 1.0 1.5 agarose
gel? Why are there varying concentrations of
agarose gels?
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Varying concentrations of agarose
Why are there varying concentrations of agarose
gels? Higher concentrations provide better
resolution for smaller DNA fragments Lower
concentrations provide better resolution for
larger DNA fragments
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Tips

• Major reason for this experiment not working is
  not having all the reaction components in the
  correct tube.
• Tick list
• Droplets on tube wall
• Master Mix

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Tick list
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Droplet method
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Droplet method

• Check pipet tip for solution.
• Eject solution on the side of tube.
• Check for a droplet.
• To get the solution in the droplet to the bottom
  of the tube tap the tube on the bench.

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Master Mix

• On the board.

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100bp Cycle
ladder 5 10 15 20 25 30