Two DNA Size Standards

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Two DNA Size Standards
One Kilobase Ladder A Synthetic Mixture of
Molecules (3 kb brightest)
Lambda DNA cut with the restriction enzyme (RE)
HindIII Amounts of DNA in different bands vary
Electrophoretic mobility (µ) 1/ log MW Use
semi-logarithmic paper to avoid converting MWs to
logs
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Semi-Logarithmic Graph
Label y axis with sizes (in kb or bp) in broad
range that includes sizes of all molecules being
analyzed
Label x axis with distance migrated in mm
10 20 30
40 50
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Your First Agarose Gel
Yeast Genomic DNA HaeIII EcoRI uncut pKAN pAMP
1Kb ? HindIII
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Electrophoretic Migration
1 Gel
MW
2 Gel
Migration Distance in mm
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Restriction Endonucleases, The Workhorses of
Molecular Biology

• REs are found in many species of bacteria
  function as primitive immune systems
• REs are endonucleases because they cleave within
  DNA molecules rather than at the ends
• REs cleave DNA at specific DNA sequences (they do
  not degrade DNA) usually 4 - 8 bp in length
• REs are used to make novel DNA molecules not
  found in nature recombinant DNA

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The gt300 Res Differ in Properties

• EcoRI GAATTC -G AATTC-
  CTTAAG -CTTAA G-
• generates cohesive ends
• HaeIII -GGCC- -GG CC-
  -CCGG- -CC GG-
• generates blunt ends

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Frequency of Cleavage Decreases as Length of
Recognition Site Increases

• EcoRI GAATTC -G AATTC-
  CTTAAG -CTTAA G-
• 6 bp recognition sites occur on average 1/4,096
  bp
• HaeIII -GGCC- -GG CC-
  -CCGG- -CC GG-
• 4 bp recognition sites occur on average 1/256 bp

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REs Cleave Complex DNAs into Smaller Fragments
for Analysis or Cloning

• Digested Genomic DNA
• Digested Plasmid DNA

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Restriction Fragment Length Polymorphisms (RFLPs)
1 kb 5 kb
1 kb 2 kb 3kb
EcoRI
EcoRI
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Using Restriction Fragment Length Polymorphisms
to Diagnose Disease
?A?A
?A?S ?S?S
5 kb 3 kb 2 kb 1 kb
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Your First Agarose Gel
Yeast Genomic DNA HaeIII EcoRI uncut pKAM pAMP
1Kb ? HindIII