Molecular Tools for Studying

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Chapter 5 Molecular Tools for Studying Genes and
Gene Activity
Jay D. Hunt, Ph.D. Department of Biochemistry and
Molecular Biology CSRB 4D1 568-4734 jhunt_at_lsuhsc.e
du
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1. Electrophoresis
2. Agarose gel electrophoresis

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• Separates DNA (or RNA or Protein) fragments on
  the basis of charge and size
• Because DNA is an acid, it looses protons in
  basic buffers thus it has a negative charge that
  is uniform per unit length
• Agarose (a polysaccharide) or other gel matrices
  are difficult for large DNA fragments to move
  through
• The larger the fragment, the more difficulty it
  has moving through gels
• By placing DNA in a gel, then applying a voltage
  across the gel, the negatively charged DNA will
  move toward the anode (positive electrode)
• Large fragments lag behind while small fragments
  move through the gel relatively rapidly

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Gel Electrophoresis
Large
Small
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Text Art Page 91
The electrophoretic mobility of a DNA fragment
is inversely proportional to the log of its size.
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Figure 5.2b
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Figure 5.1b
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• Agarose gel electrophoresis

Agarose () Standard NuSieve NuSieve 31
0.5 700 bp-25 Kbp
0.8 500 bp-15 Kbp 800 bp-10 Kbp
1.0 250 bp-12 Kbp 400 bp-8 Kbp
1.2 150 bp-6 Kbp 300 bp-7 Kbp
1.5 80 bp-4 Kbp 200 bp-4 Kbp
2.0 100 bp-3 Kbp
3.0 50 bp-1 Kbp 500 bp-1 Kbp
4.0 100 bp-500 bp
6.0 10 bp-100 bp
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1. Electrophoresis
2. Agarose gel electrophoresis
3. PFGE

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But, what if you want to separate larger
fragments, such as entire yeast chromosomes?
Pulsed-field gel electrophoresis (PFGE) can
resolve fragments from 200 Kpb (0.2 Mbp) to 6000
Kbp (6 Mbp).
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Figure 5.3
2.2 Mbp
0.2 Mbp
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1. Electrophoresis
2. Agarose gel electrophoresis
3. PFGE
4. SDS-PAGE

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• Electrophoresis of proteins using
  SDS-polyacrylamide gel electrophoresis (SDS-PAGE)
• Denaturing electrophoresis
• Detergent (SDS)
• Reducing agent (b-mercaptoethanol)
• Heat
• SDS binds to denatured proteins, making them
  negatively charged
• Migrate through gel based on size
• Molecular weight markers allow for estimation of
  size of polypeptide
• Modifications (e.g., glycosylation) can
  significantly impact the apparent size of the
  protein

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• Polyacrylamide gels are composed of chains of
  polymerized acrylamide that are cross-linked by a
  bifunctional agent
• N,N-methylene-bis-acrylamide
• Size of pores decrease as the ratio of
  bisacrylamideacrylamide increases, reaching a
  minimum at 120 ratio
• A 129 ratio is most commonly used, as it is
  capable of resolving polypeptides that differ is
  size by as little as 3

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• Electrophoresis of proteins using SDS-PAGE

Acrylamide Concentration () Linear Range of Separation (kD)
15 10-43
12 12-60
10 20-80
7.5 36-94
5.0 57-212
Molar ratio of bisacrylamideacrylamide is 129
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Figure 5.4
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1. Electrophoresis
2. Agarose gel electrophoresis
3. PFGE
4. SDS-PAGE
5. 2-D gels

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Standard SDS polyacrylamide gel
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Treat cells with a drug
Treat cells with vehicle (control)
Label proteins with Cy5 (blue)
Label proteins with Cy3 (red)
2D-gel
2D-gel
Overlay gels
Analyze for differences
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Figure 5.5
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1. Electrophoresis
2. Agarose gel electrophoresis
3. PFGE
4. SDS-PAGE
5. 2-D gels
6. Other types of chromatography
7. Ion-exchange chromatography

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Figure 5.6
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1. Electrophoresis
2. Agarose gel electrophoresis
3. PFGE
4. SDS-PAGE
5. 2-D gels
6. Other types of chromatography
7. Ion-exchange chromatography
8. Gel filtration chromatography

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Figure 5.7a
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Figure 5.7b
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1. Electrophoresis
2. Agarose gel electrophoresis
3. PFGE
4. SDS-PAGE
5. 2-D gels
6. Other types of chromatography
7. Ion-exchange chromatography
8. Gel filtration chromatography
9. Autoradiography
10. X-ray film

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Figure 5.8
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X-ray Film Cassette
Intensifying Screen b-emitters only (3H,
14C, 35S, 32P) -70C or cooler
X-ray Film
Nitrocellulose or Nylon Membrane
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Intensifying screen (fluoresces with b-rays)
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Figure 5.9
Densitometry
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1. Electrophoresis
2. Agarose gel electrophoresis
3. PFGE
4. SDS-PAGE
5. 2-D gels
6. Other types of chromatography
7. Ion-exchange chromatography
8. Gel filtration chromatography
9. Autoradiography
10. X-ray film
11. Phosphorimaging

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X-ray film cassette
Storage Phosphor plate
Nitrocellulose or Nylon Membrane
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• Phosphorimaging is much more sensitive than X-ray
  film
• lt0.95 dpm/mm2 for 1 hr exposure to 14C
• lt0.15 dpm/mm2 for 1 hr exposure to 32P
• Dynamic range of 5 orders of magnitude
• Shorter exposure times (50-90)

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Figure 5.10
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1. Electrophoresis
2. Agarose gel electrophoresis
3. PFGE
4. SDS-PAGE
5. 2-D gels
6. Other types of chromatography
7. Ion-exchange chromatography
8. Gel filtration chromatography
9. Autoradiography
10. X-ray film
11. Phosphorimaging
12. Liquid scintillation counting

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1. Electrophoresis
2. Agarose gel electrophoresis
3. PFGE
4. SDS-PAGE
5. 2-D gels
6. Other types of chromatography
7. Ion-exchange chromatography
8. Gel filtration chromatography
9. Autoradiography
10. X-ray film
11. Phosphorimaging
12. Liquid scintillation counting
13. Non-radioactive tracers

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Figure 5.11
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1. Electrophoresis
2. Other types of chromatography
3. Autoradiography
4. Nucleic acid blotting
5. Southern blotting

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Figure 5.12
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Southern blot transfer of DNA from a gel to a
solid support membrane Northern blot transfer
of RNA from a gel to a solid support membrane
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1. Electrophoresis
2. Other types of chromatography
3. Autoradiography
4. Nucleic acid blotting
5. Southern blotting
6. DNA fingerprinting

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Figure 5.13
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Figure 5.14
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Figure 5.15
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1. Electrophoresis
2. Other types of chromatography
3. Autoradiography
4. Nucleic acid blotting
5. Southern blotting
6. DNA fingerprinting
7. Northern blotting

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Figure 5.16
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1. Electrophoresis
2. Other types of chromatography
3. Autoradiography
4. Nucleic acid blotting
5. Southern blotting
6. DNA fingerprinting
7. Northern blotting
8. FISH

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Figure 5.17
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1. Electrophoresis
2. Other types of chromatography
3. Autoradiography
4. Nucleic acid blotting
5. Southern blotting
6. DNA fingerprinting
7. Northern blotting
8. FISH
9. DNA sequencing

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Figure 5.18
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Figure 5.19
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Figure 5.20a
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Figure 5.20b
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Figure 5.20c
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1. Electrophoresis
2. Other types of chromatography
3. Autoradiography
4. Nucleic acid blotting
5. Southern blotting
6. DNA fingerprinting
7. Northern blotting
8. FISH
9. DNA sequencing
10. Restriction mapping

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Figure 5.21
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Figure 5.22
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Figure 5.23
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Figure 5.24
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1. Electrophoresis
2. Other types of chromatography
3. Autoradiography
4. Nucleic acid blotting
5. Southern blotting
6. DNA fingerprinting
7. Northern blotting
8. FISH
9. DNA sequencing
10. Restriction mapping
11. Site-directed mutagenesis

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Figure 5.25
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1. Electrophoresis
2. Other types of chromatography
3. Autoradiography
4. Nucleic acid blotting
5. DNA sequencing
6. Restriction mapping
7. Site-directed mutagenesis
8. Mapping and quantifying transcripts
9. S1-nuclease mapping
10. 5-end

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Figure 5.26
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1. Electrophoresis
2. Other types of chromatography
3. Autoradiography
4. Nucleic acid blotting
5. DNA sequencing
6. Restriction mapping
7. Site-directed mutagenesis
8. Mapping and quantifying transcripts
9. S1-nuclease mapping
10. 5-end
11. 3-end

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Figure 5.27
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Figure 5.28
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Figure 5.27
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1. Electrophoresis
2. Other types of chromatography
3. Autoradiography
4. Nucleic acid blotting
5. DNA sequencing
6. Restriction mapping
7. Site-directed mutagenesis
8. Mapping and quantifying transcripts
9. S1-nuclease mapping
10. 5-end
11. 3-end
12. Primer extension

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Figure 5.29
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1. Electrophoresis
2. Other types of chromatography
3. Autoradiography
4. Nucleic acid blotting
5. DNA sequencing
6. Restriction mapping
7. Site-directed mutagenesis
8. Mapping and quantifying transcripts
9. S1-nuclease mapping
10. 5-end
11. 3-end
12. Primer extension
13. Run-off and G-less cassette assays

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Figure 5.30
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Figure 5.31
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1. Electrophoresis
2. Other types of chromatography
3. Autoradiography
4. Nucleic acid blotting
5. DNA sequencing
6. Restriction mapping
7. Site-directed mutagenesis
8. Mapping and quantifying transcripts
9. Quantifying transcription in vivo
10. Nuclear run-on transcription

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Figure 5.32
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1. Electrophoresis
2. Other types of chromatography
3. Autoradiography
4. Nucleic acid blotting
5. DNA sequencing
6. Restriction mapping
7. Site-directed mutagenesis
8. Mapping and quantifying transcripts
9. Quantifying transcription in vivo
10. Nuclear run-on transcription
11. Reporter genes

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Figure 5.33a
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Figure 5.33b
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1. Electrophoresis
2. Other types of chromatography
3. Autoradiography
4. Nucleic acid blotting
5. DNA sequencing
6. Restriction mapping
7. Site-directed mutagenesis
8. Mapping and quantifying transcripts
9. Quantifying transcription in vivo
10. DNA-Protein interactions
11. Filter binding assay

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Figure 5.34
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1. Electrophoresis
2. Other types of chromatography
3. Autoradiography
4. Nucleic acid blotting
5. DNA sequencing
6. Restriction mapping
7. Site-directed mutagenesis
8. Mapping and quantifying transcripts
9. Quantifying transcription in vivo
10. DNA-Protein interactions
11. Filter binding assay
12. Gel mobility shift assay (EMSA)

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Figure 5.35
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1. Electrophoresis
2. Other types of chromatography
3. Autoradiography
4. Nucleic acid blotting
5. DNA sequencing
6. Restriction mapping
7. Site-directed mutagenesis
8. Mapping and quantifying transcripts
9. Quantifying transcription in vivo
10. DNA-Protein interactions
11. Filter binding assay
12. Gel mobility shift assay (EMSA)
13. Footprinting

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Figure 5.36a
DNase Footprinting
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Figure 5.36b
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Figure 5.37a
DMS Footprinting
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Figure 5.37b
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1. Electrophoresis
2. Other types of chromatography
3. Autoradiography
4. Nucleic acid blotting
5. DNA sequencing
6. Restriction mapping
7. Site-directed mutagenesis
8. Mapping and quantifying transcripts
9. Quantifying transcription in vivo
10. DNA-Protein interactions
11. Knockouts

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Figure 5.38
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Figure 5.39