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 [email protected]

Chapter 5Molecular Tools for Studying

Genes and Gene ActivityJay D. Hunt, Ph.D.

Department of Biochemistry and Molecular BiologyCSRB 4D1568-4734

[email protected]

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).

+|

+ |

+|

+ |

+|

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

+|

Proteins stop migrating when they reach their isoelectric point (pH at which they have no net charge)

Isoelectric focusing

+|

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


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


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



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



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



III. AutoradiographyI. X-ray filmII. PhosphorimagingIII. Liquid scintillation counting



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. Southern blottingI. DNA fingerprinting

Figure 5.13

Figure 5.14

Figure 5.15



II. Northern blotting

Figure 5.16



II. Northern blottingIII. FISH

Figure 5.17




V. DNA sequencing

Figure 5.18

Figure 5.19

Figure 5.20a

Figure 5.20b

Figure 5.20c




V. DNA sequencingVI. Restriction mapping

Figure 5.21

Figure 5.22

Figure 5.23

Figure 5.24




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. S1-nuclease mappingI. 5’-endII. 3’-end

Figure 5.27

Use Klenow to fill in the overhangs

Figure 5.28

Figure 5.27



II. Primer extension

Figure 5.29



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. Filter binding assayII. Gel mobility shift assay (EMSA)

Figure 5.35


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

Documents

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 [email protected]