1. Molecular Biology Fourth Edition
Lecture PowerPoint to accompany
Molecular Biology Fourth Edition
Robert F. Weaver
Chapter 5
Molecular Tools for Studying Genes and Gene Activity
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.

2. 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

3. Gel Electrophoresis
Gel electrophoresis is used to separate different species of:
Nucleic acid
Protein

4. DNA Gel Electrophoresis
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

5. DNA Separation by Agarose Gel Electrophoresis
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

6. DNA Size Estimation Comparison with standards permits size estimation
Mobility of fragments are plotted v. log of molecular weight (or number of base pairs)
Electrophoresis of unknown DNA in parallel with standard fragments permits size estimation
Same principles apply to RNA separation

7. Electrophoresis of Large DNA
Special techniques are required for DNA fragments larger than about 1 kilobases
Instead of constant current, alternate long pulses of current in forward direction with shorter pulses in either opposite or sideways direction
Technique is called pulsed-field gel electrophoresis (PFGE)

8. Protein Gel Electrophoresis
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

9. Summary
DNAs, RNAs, and proteins of various masses can be separated by gel electrophoresis
Most common gel used in nucleic acid electrophoresis is agarose
Polyacrylamide is usually used in protein electrophoresis
SDS-PAGE is used to separate polypeptides according to their masses

10. Two-Dimensional Gel Electrophoresis
While SDS-PAGE gives good resolution of polypeptides, some mixtures are so complex that additional resolution is needed
Two-dimensional gel electrophoresis can be done:
(no SDS) uses 2 consecutive gels
Sequential gels with first a pH separation, then separate in a polyacrylamide gel

11. A Simple 2-D Method Run samples in 2 gels
First dimension separates using one concentration of polyacrylamide at one pH
Second dimension uses different concentration of polyacrylamide and pH
Proteins move differently at different pH values without SDS and at different acrylamide concentrations

12. 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 longer charged
Tube gel is removed and used as the sample in the second process

13. 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

14. Ion-Exchange Chromatography
Chromatography 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

15. 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

16. 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

17. 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

18. 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

19. 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

20. 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

21. 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

22. 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 that fluoresces when hit with radioactive emissions
Acts to convert invisible radioactivity into visible light

23. 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

24. Detecting Nucleic Acids With a Nonradioactive Probe

25. 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

26. Southern Blots: Identifying Specific DNA Fragments
Digests of genomic DNA are separated on agarose gel
The separated pieces are transferred to filter by diffusion, or more recently by electrophoresing the bands onto the filter
Filter is treated with alkali to denature the DNA, resulting ssDNA binds to the filter
Probe the filter using labeled cDNA

27. Southern Blots
Probe cDNA hybridizes and a band is generated corresponding to the DNA fragment of interest
Visualize bands with x-ray film or autoradiography
Multiple bands can lead to several interpretations
Multiple genes
Several restriction sites in the gene

28. DNA Fingerprinting and DNA Typing
Southern blots are used in forensic labs to identify individuals from DNA-containing materials
Minisatellite DNA is a sequence of bases repeated several times, also called DNA fingerprint
Individuals differ in the pattern of repeats of the basic sequence
Difference is large enough that 2 people have only a remote chance of having exactly the same pattern

29. DNA Fingerprinting Process really just a Southern blot
Cut the DNA under study
with restriction enzyme
Ideally cut on either side of minisatellite but not inside
Run digest on a gel and blot
Probe with labeled
minisatellite DNA and imaged
Real samples result in very complex patterns

30. Forensic Uses of DNA Fingerprinting and DNA Typing
While people have different DNA fingerprints, parts of the pattern are inherited in a Mendelian fashion
Can be used to establish parentage
Potential to identify criminals
Remove innocent people from suspicion
Actual pattern has so many bands they can smear together indistinguishably
Forensics uses probes for just a single locus
Set of probes gives a set of simple patterns

31. 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
Spread chromosomes from a cell
Partially denature DNA creating single-stranded regions to hybridize to labeled probe
Stain chromosomes and detect presence of label on particular chromosome
Probe can be detected with a fluorescent antibody in a technique called fluorescence in situ hybridization (FISH)

32. Immunoblots
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

33. Western Blots

34. DNA Sequencing
Sanger, Maxam, Gilbert developed 2 methods for determining the exact base sequence of a cloned piece of DNA
Modern DNA sequencing is based on the Sanger method

35. Sanger Manual Sequencing
Sanger DNA sequencing method uses dideoxy nucleotides to terminate DNA synthesis
The process yields a series of DNA fragments whose size is measured by electrophoresis
Last base in each fragment is known as that dideoxy nucleotide was used to terminate the reaction
Ordering the fragments by size tells the base sequence of the DNA

36. Sanger DNA Sequencing

37. 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

38. Automated DNA Sequencing

39. Restriction Mapping
Prior to start of large-scale sequencing preliminary work is done to locate landmarks
A map based on physical characteristics is called a physical map
If restriction sites are the only map features then a restriction map has been prepared
Consider a 1.6 kb piece of DNA as an example

40. Restriction Map Example
Cut separate samples of the original 1.6 kb fragment with different restriction enzymes
Separate the digests on an agarose gel to determine the size of pieces from each digest
Can also use same digest to find the orientation of an insert cloned into a vector

41. Mapping Experiment

42. Using Restriction Mapping With an Unknown DNA Sample

43. Mapping the Unknown

44. Southern Blots and Restriction Mapping

45. Summary
Physical map tells about the spatial arrangement of physical “landmarks” such as restriction sites
In restriction mapping cut the DNA in question with 2 or more restriction enzymes in separate reactions
Measure the sizes of the resulting fragments
Cut each with another restriction enzyme and measure size of subfragments by gel electrophoresis
Sizes permit location of some restriction sites relative to others
Improve process by Southern blotting fragments and hybridizing them to labeled fragments from another restriction enzyme to reveal overlaps

46. Protein Engineering With Cloned Genes: Site-Directed Mutagenesis
Cloned genes permit biochemical microsurgery on proteins
Specific bases in a gene may be changed
Amino acids at specific sites in the protein product may also be altered
Effects of those changes on protein function can be observed
Might investigate the role of phenolic group on tyrosine compared to phenylalanine

47. Site-Directed Mutagenesis With PCR

48. Summary
Using cloned genes, can introduce changes at will to alter amino acid sequence of protein products
Mutagenized DNA can be made with:
Double-stranded DNA
Two complementary mutagenic primers
PCR
Digest the PCR product to remove wild-type DNA
Cells can be transformed with mutagenized DNA

49. 5.4 Mapping and Quantifying Transcripts
Mapping (locating start and end) and quantifying (how much transcript exists at a set time) are common procedures
Often transcripts do not have a uniform terminator, resulting in a continuum of species smeared on a gel
Techniques that specific for the sequence of interest are important

50. Northern Blots You have cloned a cDNA
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
Northern plot tells abundance of the transcript
Quantify using densitometer

51. S1 Mapping
Use S1 mapping to locate the ends of RNAs and to 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

52. S1 Mapping the 5’ End

53. S1 Mapping the 3’ End

54. Summary
In S1 mapping, a labeled DNA probe is used to detect 5’- or 3’-end of a transcript
Hybridization of the probe to the transcript protects a portion of the probe from digestion by S1 nuclease, specific for single-stranded polynucleotides
Length of the section of probe protected by the transcript locates the end of the transcript relative to the known location of an end of the probe
Amount of probe protected is proportional to concentration of transcript, so S1 mapping can be quantitative
RNase mapping uses an RNA probe and RNase

55. 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

56. Primer Extension Schematic
Start with in vivo transcription, harvest cellular RNA containing desired transcript
Hybridize labeled oligonucleotide [18nt] (primer)
Reverse transcriptase extends the primer to the 5’-end of transcript
Denature the RNA-DNA hybrid and run the mix on a high-resolution DNA gel
Can estimate transcript concentration also

57. Run-Off Transcription and G-Less Cassette Transcription
If want to assess:
Transcription accuracy
How much of this accurate transcription
Simpler method is run-off transcription
Can be used after the physiological start site is found by S1 mapping or primer extension
Useful to see effects of promoter mutation on accuracy and efficiency of transcription

58. Run-Off Transcription
DNA fragment containing gene to transcribe is cut with restriction enzyme in middle of transcription region
Transcribe the truncated fragment in vitro using labeled nucleotides, as polymerase reaches truncation it “runs off” the end
Measure length of run-off transcript compared to location of restriction site at 3’-end of truncated gene

59. G-Less Cassette Assay
Variation of the run-off technique, instead of cutting the gene with restriction enzyme, insert a stretch of nucleotides lacking guanines in nontemplate strand just downstream of promoter
As promoter is stronger a greater number of aborted transcripts is produced

60. Schematic of the G-Less Cassette Assay
Transcribe altered template in vitro with CTP, ATP and UTP one of which is labeled, but no GTP
Transcription will stop when the first G is required resulting in an aborted transcript of predictable size
Separate transcripts on a gel and measure transcription activity with autoradiography

61. Summary
Run-off transcription is a means of checking efficiency and accuracy of in vitro transcription
Gene is truncated in the middle and transcribed in vitro in presence of labeled nucleotides
RNA polymerase runs off the end making an incomplete transcript
Size of run-off transcript locates transcription start site
Amount of transcript reflects efficiency of transcription
In G-less cassette transcription, a promoter is fused to dsDNA cassette lacking Gs in nontemplate strand
Construct is transcribed in vitro in absence of of GTP
Transcription aborts at end of cassette for a predictable size band on a gel

62. 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
These techniques do not really reveal the rate of transcript synthesis as concentration involves both:
Transcript synthesis
Transcript degradation

63. Nuclear Run-On Transcription
Isolate nuclei from cells, allow them to extend in vitro the transcripts already started in vivo in a technique called run-on transcription
RNA polymerase that has already initiated transcription will “run-on” or continue to elongate same RNA chains
Effective as initiation of new RNA chains in isolated nuclei does not generally occur

64. Run-On Analysis
Results will show transcription rates and an idea of which genes are transcribed
Identification of labeled run-on transcripts is best done by dot blotting
Spot denatured DNAs on a filter
Hybridize to labeled run-on RNA
Identify the RNA by DNA to which it hybridizes
Conditions of run-on reaction can be manipulated with effects of product can be measured

65. Nuclear Run-On Transcription Diagram

66. Reporter Gene Transcription
Place a surrogate reporter gene under control of a specific promoter, measure accumulation of product of this reporter gene
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

67. Measuring Protein Accumulation in Vivo
Gene activity can be monitored by measuring the accumulation of protein (the ultimate gene product)
Two primary methods of measuring protein accumulation
Immunoblotting / Western blotting
Immunoprecipitation

68. 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

69. 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
Which bases of DNA interact with a protein

70. 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

71. Nitrocellulose Filter-Binding Assay
dsDNA is labeled and mixed with protein
Pour dsDNA through a nitrocellulose filter
Measure amount of radioactivity that passed through filter and retained on filter

72. Gel Mobility Shift
DNA moves through a gel faster when it is not bound to protein
Gel shift assays detect interaction between protein and DNA by reduction of the electrophoretic mobility of a small DNA bound to a protein

73. 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

74. DNase Footprinting
Protein binding to DNA covers the binding site and protects from attack by DNase
End label DNA, 1 strand only
Protein binds DNA
Treat complex with DNase I
mild conditions for average
of 1 cut per molecule
Remove protein from DNA,
separate strands and run on
a high-resolution
polyacrylamide gel

75. DMS Footprinting
Dimethylsulfate (DMS) is a methylating agent which can fit into DNA nooks and crannies
Starts as DNase, then methylate with DMS at conditions for 1 methylation per DNA molecule

76. Summary
Footprinting finds target DNA sequence or binding site of a DNA-binding protein
DNase footprinting binds protein to end-labeled DNA target, then attacks DNA-protein complex with DNase
DNA fragments are electrophoresed with protein binding site appearing as a gap in the pattern where protein protected DNA from degradation
DMS, DNA methylating agent is used to attack the DNA-protein complex
Hydroxyl radicals – copper- or iron-containing organometallic complexes generate hydroxyl radicals that break the DNA strands

77. 5.7 Finding RNA Sequences That Interact With Other Molecules
SELEX is systematic evolution of ligands by exponential enrichment
SELEX is a method to find RNA sequences that interact with other molecules, even proteins
RNAs that interact with a target molecule are selected by affinity chromatography
Convert to dsDNA and amplify by PCR
RNAs are now highly enriched for sequences that bind to the target molecule

78. Functional SELEX
Functional SELEX is a variation where the desired function alters RNA so it can be amplified
If desired function is enzymatic, mutagenesis can be introduced into the amplification step to produce variants with higher activity

79. 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

80. Stage 1 of the Knockout Mouse
Cloned DNA containing the mouse gene to be knocked out is interrupted with another gene that confers resistance to neomycin
A thymidine kinase gene is placed outside the target gene
Mix engineered mouse DNA with stem cells so interrupted gene will find way into nucleus and homologous recombination with altered gene and resident, intact gene
These events are rare, many cells will need to be screened using the introduced genes

81. Making a Knockout Mouse: Stage 1

82. Stage 2 of the Knockout Mouse
Introduce the interrupted gene into a whole mouse
Inject engineered cells into a mouse blastocyst
Embryo into a surrogate mother who gives birth to chimeric mouse with patchy coat
True heterozygote results when chimera mates with a black mouse to produce brown mice, half of which will have interrupted gene

83. Making a Knockout Mousse: Stage 2

84. Knockout Results
Phenotype may not be obvious in the progeny, but still instructive
Other cases can be lethal with the mice dying before birth
Intermediate effects are also common and may require monitoring during the life of the mouse