Chapter 5Molecular Tools for Studying
Genes and Gene ActivityJay D. Hunt, Ph.D.
Department of Biochemistry and Molecular BiologyCSRB 4D1568-4734
I. ElectrophoresisI. Agarose gel electrophoresis
• 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
Gel Electrophoresis
+
-
Direction of
DNATravel
Wells
Small
Large
Text Art Page 91
The electrophoretic mobility of a DNA fragment is inversely proportional to the log of its size.
Figure 5.2b
1020
3040
5060
70
3 mm
Figure 5.1b
• Agarose gel electrophoresis
Agarose (%) Standard NuSieve NuSieve 3:1
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
I. ElectrophoresisI. Agarose gel electrophoresisII. PFGE
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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+ |
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+ |
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Figure 5.3
2.2 Mbp
0.2 Mbp
I. ElectrophoresisI. Agarose gel electrophoresisII. PFGEIII. SDS-PAGE
• Electrophoresis of proteins using SDS-polyacrylamide gel electrophoresis (SDS-PAGE)– Denaturing electrophoresis
• Detergent (SDS)• Reducing agent (-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
• 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
bisacrylamide:acrylamide increases, reaching a minimum at ~1:20 ratio
– A 1:29 ratio is most commonly used, as it is capable of resolving polypeptides that differ is size by as little as 3%
•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 bisacrylamide:acrylamide is 1:29
Figure 5.4
I. ElectrophoresisI. Agarose gel electrophoresisII. PFGEIII. SDS-PAGEIV. 2-D gels
Acidic
Basic
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Proteins stop migrating when they reach their isoelectric point (pH at which they have no net charge)
Isoelectric focusing
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Standard SDS polyacrylamide gel
Separated by isoelectric point
Separated by Size
Treat cells with a drug Treat cells with vehicle (control)
Label proteins with Cy3 (red) Label proteins with Cy5 (blue)
2D-gel 2D-gel
Overlay gels
Analyze for differences
Figure 5.5
I. ElectrophoresisI. Agarose gel electrophoresisII. PFGEIII. SDS-PAGEIV. 2-D gels
II. Other types of chromatographyI. Ion-exchange chromatography
DEAE-Sephadex(Positively Charged)
Negatively charged proteins bind to resin (the stronger the charge, the tighter the binding)
LowSalt
HighSalt
Weakest bound protein (weakest negative charge) comes off first.
Tighter bound protein elutes with higher concentrations of salt
Figure 5.6
I. ElectrophoresisI. Agarose gel electrophoresisII. PFGEIII. SDS-PAGEIV. 2-D gels
II. Other types of chromatographyI. Ion-exchange chromatographyII. Gel filtration chromatography
Figure 5.7a
Sephadex
Largest proteins come off first (void volume).
Smaller proteins elute with additional buffer
Buffer
Figure 5.7b
I. ElectrophoresisI. Agarose gel electrophoresisII. PFGEIII. SDS-PAGEIV. 2-D gels
II. Other types of chromatographyI. Ion-exchange chromatographyII. Gel filtration chromatography
III. AutoradiographyI. X-ray film
Figure 5.8
X-ray Film Cassette
Nitrocellulose or Nylon MembraneX-ray Film
Intensifying Screen:•-emitters only•(3H, 14C, 35S, 32P)•-70°C or cooler
Ag Ag Ag Ag Ag Ag Ag Ag Ag Ag Ag Ag
Intensifying screen (fluoresces with -rays)
32P 32P
Figure 5.9
Densitometry
I. ElectrophoresisI. Agarose gel electrophoresisII. PFGEIII. SDS-PAGEIV. 2-D gels
II. Other types of chromatographyI. Ion-exchange chromatographyII. Gel filtration chromatography
III. AutoradiographyI. X-ray filmII. Phosphorimaging
Nitrocellulose or Nylon Membrane
Storage Phosphor plate
X-ray film cassette
•Phosphorimaging is much more sensitive than X-ray film•<0.95 dpm/mm2 for 1 hr exposure to 14C•<0.15 dpm/mm2 for 1 hr exposure to 32P
•Dynamic range of 5 orders of magnitude•Shorter exposure times (50-90%)
Figure 5.10
I. ElectrophoresisI. Agarose gel electrophoresisII. PFGEIII. SDS-PAGEIV. 2-D gels
II. Other types of chromatographyI. Ion-exchange chromatographyII. Gel filtration chromatography
III. AutoradiographyI. X-ray filmII. PhosphorimagingIII. Liquid scintillation counting
I. ElectrophoresisI. Agarose gel electrophoresisII. PFGEIII. SDS-PAGEIV. 2-D gels
II. Other types of chromatographyI. Ion-exchange chromatographyII. Gel filtration chromatography
III. AutoradiographyI. X-ray filmII. PhosphorimagingIII. Liquid scintillation countingIV. Non-radioactive tracers
Figure 5.11
I. ElectrophoresisII. Other types of chromatographyIII. AutoradiographyIV. Nucleic acid blotting
I. Southern blotting
Figure 5.12
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
I. ElectrophoresisII. Other types of chromatographyIII. AutoradiographyIV. Nucleic acid blotting
I. Southern blottingI. DNA fingerprinting
Figure 5.13
Figure 5.14
Figure 5.15
I. ElectrophoresisII. Other types of chromatographyIII. AutoradiographyIV. Nucleic acid blotting
I. Southern blottingI. DNA fingerprinting
II. Northern blotting
Figure 5.16
I. ElectrophoresisII. Other types of chromatographyIII. AutoradiographyIV. Nucleic acid blotting
I. Southern blottingI. DNA fingerprinting
II. Northern blottingIII. FISH
Figure 5.17
I. ElectrophoresisII. Other types of chromatographyIII. AutoradiographyIV. Nucleic acid blotting
I. Southern blottingI. DNA fingerprinting
II. Northern blottingIII. FISH
V. DNA sequencing
Figure 5.18
Figure 5.19
Figure 5.20a
Figure 5.20b
Figure 5.20c
I. ElectrophoresisII. Other types of chromatographyIII. AutoradiographyIV. Nucleic acid blotting
I. Southern blottingI. DNA fingerprinting
II. Northern blottingIII. FISH
V. DNA sequencingVI. Restriction mapping
Figure 5.21
Figure 5.22
Figure 5.23
Figure 5.24
I. ElectrophoresisII. Other types of chromatographyIII. AutoradiographyIV. Nucleic acid blotting
I. Southern blottingI. DNA fingerprinting
II. Northern blottingIII. FISH
V. DNA sequencingVI. Restriction mappingVII.Site-directed mutagenesis
Figure 5.25
I. ElectrophoresisII. Other types of chromatographyIII. AutoradiographyIV. Nucleic acid blottingV. DNA sequencingVI. Restriction mappingVII.Site-directed mutagenesisVIII.Mapping and quantifying transcripts
I. S1-nuclease mappingI. 5’-end
Figure 5.26
Polynucleotide kinase labels at 5’ end
I. ElectrophoresisII. Other types of chromatographyIII. AutoradiographyIV. Nucleic acid blottingV. DNA sequencingVI. Restriction mappingVII.Site-directed mutagenesisVIII.Mapping and quantifying transcripts
I. S1-nuclease mappingI. 5’-endII. 3’-end
Figure 5.27
Use Klenow to fill in the overhangs
Figure 5.28
Figure 5.27
I. ElectrophoresisII. Other types of chromatographyIII. AutoradiographyIV. Nucleic acid blottingV. DNA sequencingVI. Restriction mappingVII.Site-directed mutagenesisVIII.Mapping and quantifying transcripts
I. S1-nuclease mappingI. 5’-endII. 3’-end
II. Primer extension
Figure 5.29
I. ElectrophoresisII. Other types of chromatographyIII. AutoradiographyIV. Nucleic acid blottingV. DNA sequencingVI. Restriction mappingVII.Site-directed mutagenesisVIII.Mapping and quantifying transcripts
I. S1-nuclease mappingI. 5’-endII. 3’-end
II. Primer extensionIII. Run-off and G-less cassette assays
Figure 5.30
Figure 5.31
I. ElectrophoresisII. Other types of chromatographyIII. AutoradiographyIV. Nucleic acid blottingV. DNA sequencingVI. Restriction mappingVII.Site-directed mutagenesisVIII.Mapping and quantifying transcriptsIX. Quantifying transcription in vivo
I. Nuclear run-on transcription
Figure 5.32
I. ElectrophoresisII. Other types of chromatographyIII. AutoradiographyIV. Nucleic acid blottingV. DNA sequencingVI. Restriction mappingVII.Site-directed mutagenesisVIII.Mapping and quantifying transcriptsIX. Quantifying transcription in vivo
I. Nuclear run-on transcriptionII. Reporter genes
Figure 5.33a
Figure 5.33b
I. ElectrophoresisII. Other types of chromatographyIII. AutoradiographyIV. Nucleic acid blottingV. DNA sequencingVI. Restriction mappingVII.Site-directed mutagenesisVIII.Mapping and quantifying transcriptsIX. Quantifying transcription in vivoX. DNA-Protein interactions
I. Filter binding assay
Figure 5.34
I. ElectrophoresisII. Other types of chromatographyIII. AutoradiographyIV. Nucleic acid blottingV. DNA sequencingVI. Restriction mappingVII.Site-directed mutagenesisVIII.Mapping and quantifying transcriptsIX. Quantifying transcription in vivoX. DNA-Protein interactions
I. Filter binding assayII. Gel mobility shift assay (EMSA)
Figure 5.35
I. ElectrophoresisII. Other types of chromatographyIII. AutoradiographyIV. Nucleic acid blottingV. DNA sequencingVI. Restriction mappingVII.Site-directed mutagenesisVIII.Mapping and quantifying transcriptsIX. Quantifying transcription in vivoX. DNA-Protein interactions
I. Filter binding assayII. Gel mobility shift assay (EMSA)III. Footprinting
Figure 5.36a
DNase Footprinting
Figure 5.36b
Figure 5.37a
DMS Footprinting
Figure 5.37b
I. ElectrophoresisII. Other types of chromatographyIII. AutoradiographyIV. Nucleic acid blottingV. DNA sequencingVI. Restriction mappingVII.Site-directed mutagenesisVIII.Mapping and quantifying transcriptsIX. Quantifying transcription in vivoX. DNA-Protein interactionsXI. Knockouts
Figure 5.38
Figure 5.39