1. Chapter 5 Molecular Tools for Studying Genes and Gene Activity
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2. Chapter Outline Molecular Separations Labeled Tracers
Gel Electrophoresis
Two-Dimensional Gel Electrophoresis
Ion-Exchange Chromatography
Gel filtration Chromatography
Affinity Chromatography
Labeled Tracers
Autoradiography
Phosphorimaging
Liquid Scintillation Counting
Nonradioactive Tracers
Using Nucleic Acid Hybridization
Southern Blots: Identifying Specific DNA Fragments
DNA Fingerprinting and DNA Typing
Forensic Uses of DNA Fingerprinting and DNA Typing
Immunoblots (Western blots)
DNA sequencing
Restriction Mapping
Protein Engineering with Cloned Genes: Site-Directed Mutagenesis

3. Chapter Outline Mapping and Quantifying Transcripts
Northern Blots
S1 Mapping
Primer extension
Run-off Transcription and G-Less Cassette Transcription
Measuring Transcription Rates in vivo
Assay DNA-Protein Interaction
Finding RNA sequences that interact with other molecule
Knockouts

4. 5.1 Molecular Separations
Often mixtures of proteins or nucleic acids are generated during the course of molecular biological procedures
A protein may need to be purified from a crude cellular extract
A particular nucleic acid molecule made in a reaction needs to be purified

5. 5.1 Molecular Separations
Gel Electrophoresis
Gel electrophoresis is used to separate different species of:
Nucleic acid
Protein

6. DNA Gel Electrophoresis
Slots / wells
Horizontal
agarose gel
DNA
Melted agarose is poured into a form equipped with removable comb
Comb “teeth” form slots in the solidified agarose
DNA samples are placed in the slots
An electric current is run through the gel at a neutral pH
+++++
Dye:
fluoresces UV

7. DNA Separation by Agarose Gel Electrophoresis
Large
DNA is negatively charged due to phosphates in its backbone and moves to anode, the positive pole
Small DNA pieces have little frictional drag so move rapidly
Large DNAs have more frictional drag so their mobility is slower
Result distributes DNA according to size
Largest near the top
Smallest near the bottom
DNA is stained with fluorescent dye
Small

8. pulsed-field gel electrophoresis (PFGE)
Separate DNA up to Megabases (106bps)
long pulses of current in forward direction with shorter pulses in either opposite or sideways direction
2.2Mb
Dye:
Ethidium
Bromide
0.2Mb

9. Protein Gel Electrophoresis
Mr (kD)
Separation of proteins is done using a gel made of polyacrylamide (polyacrylamide gel electrophoresis = PAGE)
Treat proteins to denature subunits with detergent such as SDS
SDS coats polypeptides with negative charges so all move to anode
Masks natural charges of protein subunits so all move relative to mass not charge
As with DNA smaller proteins move faster toward the anode

10. Two-Dimensional Gel Electrophoresis Details
A two process method:
Isoelectric focusing gel: mixture of proteins electrophoresed through gel in a narrow tube containing a pH gradient
Negatively charged protein moves to its isoelectric point at which it is no net charged
Tube gel is removed and used as the sample in the second process

11. More Two-Dimensional Gel Electrophoresis Details
Standard SDS-PAGE:
Tube gel is removed and used as the sample at the top of a standard polyacrylamide gel
Proteins partially resolved by isoelectric focusing are further resolved according to size
When used to a compare complex mixtures of proteins prepared under two different conditions, even subtle differences are visible
IEF
SDS-PAGE
pH gradient
      
Proteomics
size

12. Benzoic acid (-)
Red
Benzoic acid (+)
Blue
Fig5.5 Two-Dimensional gel electrophoresis

13. Ion-Exchange Chromatography
Chromatography 層析originally referred to the pattern seen after separating colored substances on paper
Ion-exchange chromatography uses a resin to separate substances by charge
This is especially useful for proteins
Resin is placed in a column and sample loaded onto the column material

14. Separation by Ion-Exchange Chromatography
Once the sample is loaded buffer is passed over the resin + sample
As ionic strength of elution buffer increases, samples of solution flowing through the column are collected
Samples are tested for the presence of the protein of interest

15. Gel Filtration Chromatography
Protein size is a valuable property that can be used as a basis of physical separation
Gel filtration uses columns filled with porous resins that let in smaller substances, exclude larger ones
Larger substances travel faster through the column
[Large Molecule]
Whiffle Ball
[small Molecule]

16. Affinity Chromatography
In affinity chromatography, the resin contains a substance to which the molecule of interest has a strong and specific affinity
The molecule binds to a column resin coupled to the affinity reagent
Molecule of interest is retained
Most other molecules flow through without binding
Last, the molecule of interest is eluted from the column using a specific solution that disrupts the specific binding

17. 5.2 Labeled Tracers
For many years “labeled” has been synonymous with “radioactive”
Radioactive tracers allow vanishingly small quantities of substances to be detected
Molecular biology experiments typically require detection of extremely small amounts of a particular substance

18. Autoradiography ** ** ** *
Autoradiography is a means of detecting radioactive compounds with a photographic emulsion
Preferred emulsion is x-ray film
DNA is separated on a gel and radiolabeled
Gel is placed in contact with x-ray film for hours or days
Radioactive emissions from the labeled DNA expose the film
Developed film shows dark bands *
x-ray film
cassette
Intensifying
screen
Intensifying screen
High-energy electrons strike the screen, cause fluorescence, detected by the film

19. Autoradiography Analysis
Relative quantity of radioactivity can be assessed looking at the developed film
More precise measurements are made using densitometer
Area under peaks on a tracing by a scanner
Proportional to darkness of the bands on autoradiogram

20. Phosphorimaging
This technique is more accurate in quantifying amount of radioactivity in a substance
Response to radioactivity is much more linear
Place gel with radioactive bands in contact with a phosphorimager plate
Plate absorbs b electrons that excite molecules on the plate which remain excited until plate is scanned
Molecular excitation is monitored by a detector
Yellow<purple<magenta<light blue<green<dark blue<black (highest)

21. Liquid Scintillation Counting
Radioactive emissions from a sample create photons of visible light are detected by a photomultiplier tube in the process of liquid scintillation counting
Remove the radioactive material (band from gel) to a vial containing scintillation fluid
Fluid contains a fluor 螢石 that fluoresces when hit with radioactive emissions
Acts to convert invisible radioactivity into visible light
Counts per minute (cpm)
Smear Test

22. Nonradioactive Tracers
Newer nonradioactive tracers now rival older radioactive tracers in sensitivity
These tracers do not have hazards:
Health exposure
Handling
Disposal
Increased sensitivity is from use of a multiplier effect of an enzyme that is coupled to probe for molecule of interest

23. Detecting Nucleic Acids With a Nonradioactive Probe
Indirect
DIG(Roche)
Digoxigenin
Anti-DIG-AP

24. 5.3 Using Nucleic Acid Hybridization
Hybridization is the ability of one single-stranded nucleic acid to form a double helix with another single strand of complementary base sequence
Previous discussion focused on colony and plaque hybridization
This section looks at techniques for isolated nucleic acids

25. Southern Blots Identifying Specific DNA Fragments alkali sol.
ssDNA binds to the filter

26. Isotope label or
Nonisotope label probe

27. DNA Fingerprinting Minisatellite DNA is a sequence of bases
the ministatellite DNAs do not contain HaeIII recognition site
Minisatellite DNA
is a sequence of bases
repeated several times
DNA fingerprint
Probe with labeled
minisatellite DNA
Individuals differ in the pattern
of repeat of the basic sequence

28. No two pattern
are identical
Can be used to establish parentage
Potential to identify criminals
Remove innocent people from suspicion
Monozygotic twins
Fig 5.14 DNA fingerprint

29. In Situ Hybridization: Locating Genes in Chromosomes
Labeled probes can be used to hybridize to chromosomes and reveal which chromosome contains the gene of interest
Probe can be detected with a fluorescent antibody in a technique called fluorescence in situ hybridization (FISH)
Probe: muscle glycogen phosphorylase gene
label with dinitrophenol
Chromosome: Propidium iodide (PI) stain

30. Immunoblots (Western blots)
Immunoblots (also called Western blots) use a similar process to Southern blots
Electrophoresis of proteins
Blot the proteins from the gel to a membrane
Detect the protein using antibody or antiserum to the target protein
Labeled secondary antibody is used to bind the first antibody and increase the signal
gel
membrane

31. Sanger DNA Sequencing Deoxy nucleotide Dideoxy nucleotide
(chain terminator)
Primer ddTCP
dATP,dCTP,dGTP
DNA polymerase

32. Automated DNA Sequencing
Manual sequencing is powerful but slow
Automated sequencing uses dideoxynucleotides tagged with different fluorescent molecules
Products of each dideoxynucleotide will fluoresce a different color
Four reactions are completed, then mixed together and run out on one lane of a gel

33. Automated DNA Sequencing
dideoxynucleotides tagged
with different fluorescent molecules
Autometed sequencers (sequenators)

34. Restriction Mapping
Determine the orientation of the HindIII fragment in a cloning vector.

36. HindIII
11kb
7kb
HindIII
4kb
5kb
4kb
5kb
PstI
7kb
PstI
7kb
1kb
PstI
7kb
1kb
11kb
HindIII
11kb
7kb
HindIII
5kb
7kb
5kb
7kb
PstI
PstI
1kb
4kb
1kb
PstI
4kb

37. Southern Blots and Restriction Mapping

38. Protein Engineering With Cloned Genes: Site-Directed Mutagenesis

39. Site-Directed Mutagenesis With PCR
Protein Engineering With Cloned Genes: Site-Directed Mutagenesis
Heating
Annealing
25bp
Site-Directed Mutagenesis With PCR
TACTTC

40. 5.4 Mapping and Quantifying Transcripts
Mapping: locating start and end
quantifying: how much transcript exists at a set time

41. Northern Blots
How actively is the corresponding gene expressed in different tissues?
Find out using a Northern Blot
Obtain RNA from different tissues
Run RNA on agarose gel and blot to membrane
Hybridize to a labeled cDNA probe
Probe:G3PDH
G3PDH gene expression

42. S1 Mapping S1 mapping: 1. locate the 5’ and 3’ ends of RNAs
2. determine the amount of a given RNA in cells
at a given time
Label a ssDNA probe that can only hybridize to transcript of interest
Probe must span the sequence start to finish
After hybridization, treat with S1 nuclease which degrades ssDNA and RNA
Transcript protects part of the probe from degradation
Size of protected area can be measured by gel electrophoresis

43. S1 Mapping the 5’ End * * ? Transcription initiation site
Remove unlabel phosphate ? * *
DNA probeRNA probe
S1 nucleaseRNase
RNase
mapping
Digest single-strand
DNA and RNA

44. S1 Mapping the 3’ End ?
End-filling

45. Primer Extension
Primer extension works to determine exactly the 5’-end of a transcript to one-nucleotide accuracy
Specificity of this method is due to complementarity between primer and transcript
S1 mapping will give similar results but limits:
S1 will “nibble” ends of RNA-DNA hybrid
Also can “nibble” A-T rich regions that have melted
Might not completely digest single-stranded regions

46. Primer Extension Schematic
works to determine exactly the 5’-end of a transcript to one-nucleotide accuracy
sequencing
TTCGACTGACAGT

47. 5.5 Measuring Transcription Rates in Vivo
Primer extension, S1 mapping and Northern blotting will determine the concentration of specific transcripts at a given time
Transcript synthesis
Transcript degradation
The rate of transcript synthesis
Nuclear Run-On Transcription
Reporter Gene Expression
(END POINT)

48. Nuclear Run-On Transcription Diagram
In vitro
In vivo
To eliminate the possibility of initiation of new RNA  heparin
(binds free RNA pol.)
Nuclear Run-On Transcription Diagram

49. Reporter Gene Transcription
Reporter genes are carefully chosen to have products very convenient to assay
lacZ produces b-galactosidase which has a blue cleavage product
cat produces chloramphenicol acetyl transferase (CAT) which inhibits bacterial growth
Luciferase produces chemiluminescent compound that emits light

51. Measuring Protein Accumulation in Vivo
Gene activity can be monitored by measuring the accumulation of protein
Two primary methods of measuring protein accumulation
Immunoblotting / Western blotting
Immunoprecipitation
Label proteins by growing cells with 35S-labeled amino acid
Bind protein of interest to an antibody
Precipitate the protein-antibody complex with a secondary antibody complexed to Protein A on resin beads using a low-speed centrifuge
Determine protein level with liquid scintillation counting

52. 5.6 Assaying DNA-Protein Interactions
Study of DNA-protein interactions is of significant interest to molecular biologists
Types of interactions often studied:
Protein-DNA binding
Filter Binding
Gel Mobility Shift
Which bases of DNA interact with a protein
DNase Footprinting
DMA footprinting and Footprinting Meyhods

53. Filter Binding
Filter binding to measure DNA-protein interaction is based on the fact that double-stranded DNA will not bind by itself to a filter, but a protein-DNA complex will
Double-stranded DNA can be labeled and mixed with protein
Assay protein-DNA binding by measuring the amount of label retained on the filter

54. Nitrocellulose Filter-Binding Assay* * * *
Nitrocellulose *
No DNA bind *
ss DNA binds tightly

55. Gel Mobility Shift assay [electrophoretic mobility shift assay (EMSA)]

56. Footprinting
Footprinting shows where a target lies on DNA and which bases are involved in protein binding
Three methods are very popular:
DNase footprinting
Dimethylsulfate footprinting
Hydroxyl radical footprinting

57. DNase Footprinting 1 strand only End label DNA Remove protein from DNA
run on a high-
resolution
polyacrylamide gel

58. DMS Footprinting 1 strand only
Dimethylsulfate (DMS) is a methylating agent which can fit into DNA nooks and crannies
End label DNA
Treat with piperidine
Remove methylated purines
apurine site
DNA break

59. 5.8 Knockouts
Probing structures and activities of genes does not answer questions about the role of the gene in the life of the organism
Targeted disruption of genes is now possible in several organisms
When genes are disrupted in mice the products are called knockout mice

60. Making a Knockout Mouse: Stage 1

61. Making a Knockout Mousse: Stage 2