1. Molecular Biology Fifth Edition
Lecture PowerPoint to accompany
Molecular Biology Fifth 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
Gel electrophoresis is used to separate different species of:
Nucleic acid
Protein

3. 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 to allow the sample to travel through the gel matrix

4. DNA Separation by Agarose Gel Electrophoresis
DNA is negatively charged due to the phosphates in its backbone and moves toward the positive pole
Small DNA pieces have little frictional drag so they move rapidly
Large DNAs have more frictional drag so their mobility is slower
Distributes DNA according to size
Largest near the top
Smallest near the bottom
DNA is stained with fluorescent dye that intercalates between the bases

5. DNA 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 upon comparison
Same principles apply to RNA separation

6. 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)

7. Protein Gel Electrophoresis
Separation of proteins is done using polyacrylamide gel electrophoresis (PAGE)
Treat proteins to denature subunits with detergent such as sodium dodecyl sulfate (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

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

9. 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:
Nondenaturing gel electrophoresis (no SDS) uses 2 consecutive gels each in a different dimension
Sequential gels with distinct pH separation and polyacrylamide gel concentration

10. A Simple 2-D Method Samples are run 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

11. A More Powerful Two-Dimensional Gel Electrophoresis Technique
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

12. A More Powerful Two-Dimensional Gel Electrophoresis Technique continued
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

13. 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 the sample is 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 and exclude larger substances
As a result larger substances travel faster through the column

16. 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 their specific binding

17. Summary
High-resolution separation of proteins can be achieved by two-dimensional gel electrophoresis
Ion-exchange chromatography can be used to separate substances according to their sizes
Gel filtration chromatography uses columns filled with porous resins that let in smaller substances but exclude larger ones
Affinity chromatography is a powerful purification technique that exploits an affinity reagent with strong and specific affinity for a molecule of interest

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 a 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 a gel
The separated pieces are transferred to filter (nitrocellulose) by diffusion, or more recently by electrophoresing the DNA onto the filter
The filter is then treated with alkali to denature the DNA, resulting ssDNA binds to the filter
A labeled cDNA probe that is complementary to the DNA of interest is then applied to the filter
A positive band should be detectable where hybridization between the probe and DNA occurred

27. Southern Blots
The probe 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 a DNA fingerprint
Individuals differ in the pattern of repeats of the basic sequence
The difference is large enough that 2 people have only a remote chance of having exactly the same pattern of repeats

29. DNA Fingerprinting Process is a Southern blot Cut the DNA under study
with restriction enzyme
Ideally cut on either side of minisatellite but not inside
Run the digested DNA on a gel and blot
Probe with labeled
minisatellite DNA and image
Note that 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 for visualization and to increase the signal

33. Summary
Labeled DNA (or RNA) probes can be used to hybridize to DNAs of the same or very similar sequence on a Southern blot
DNA fingerprinting can be used as a forensic tool or to test parentage
In situ hybridization can be used to locate genes or other specific DNA sequences on whole chromosomes
Proteins can be detected and quantified in a complex mixture using Western blots

34. 5.4 DNA Sequencing
Sanger, Maxam, and 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 and uses dideoxy nucleotides to terminate DNA synthesis
The process yields a series of DNA fragments whose size is measured by electrophoresis
The 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

35. Sanger Method of DNA Sequencing

36. 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

37. High Throughput Sequencing
Once an organism’s genome sequence is known, very rapid sequencing techniques can be applied to sequence the genome of another member of the same species
Produces relatively short reads or contiguous sequences (25-35bp or bp, depending on the method) that can easily be pieced together if a reference sequence is available

38. High Throughput Sequencing
Pyrosequencing is one example that is an automated system with the advantages of speed and accuracy
- nucleotides are added one by one and the incorporation of a nucleotide is detected by a release of pyrophosphate, which leads to a flash of light
Another method (Illumina company) starts by attaching short pieces of DNA to a solid surface, amplifying each DNA in a tiny patch on the surface, then sequencing the patches together by extending them one nucleotide at a time using fluorescent chain-terminating nucleotides, whose fluoresce reveals their identity

39. Restriction Mapping
Prior to the 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

40. Restriction Map Example
Consider a 1.6 kb piece of DNA as an 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. Southern Blots and Restriction Mapping

42. Summary
Physical maps tell 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

43. 5.5 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 be altered as a result
Effects of those changes on protein function can be observed

44. PCR-based Site-Directed Mutagenesis

45. Summary
Using cloned genes, one can introduce changes that may alter the amino acid sequence of the corresponding 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

46. 5.6 Mapping and Quantifying Transcripts
In the field of molecular biology mapping (locating start and end) and quantifying (how much transcript exists at a set time) transcripts are common procedures
Often transcripts do not have a uniform terminator, resulting in a continuum of species smeared on a gel
Techniques that are specific for the sequence of interest are important

47. Northern Blots Northern blots detect RNA
Example: You have cloned a cDNA
Question: How actively is the corresponding gene expressed in different tissues?
Answer: 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

48. 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

49. S1 Mapping the 5’ End

50. S1 Mapping the 3’ End

51. Summary
A Northern blot is similar to a Southern blot but is a method used for detection of RNA
In S1 mapping, a labeled DNA probe is used to detect 5’- or 3’-end of a transcript
Amount of probe protected is proportional to concentration of transcript, so S1 mapping can be quantitative
RNase mapping is a variation on SI mapping that uses an RNA probe and RNase

52. Primer Extension Schematic
Primer extension works to determine the 5’-end of a transcript to one-nucleotide accuracy
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
- Specificity of this method is due to complementarity between primer and transcript
- S1 mapping will give similar results but with 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

53. Run-Off Transcription
A good assay to measure the rate of in vitro 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

54. Schematic of the G-Less Cassette Assay
A variation of the run-off technique in which a stretch of nucleotides lacking guanines is inserted into the nontemplate strand just downstream of the promoter
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
- The stronger the promoter, the greater number of aborted transcripts is produced

55. 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

56. 5.7 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

57. Nuclear Run-On Transcription
The idea of this assay is to isolate nuclei from cells, allow them to extend in vitro the transcripts already started in vivo
RNA polymerase that has already initiated transcription will “run-on” or continue to elongate the same RNA chains
Effective as initiation of new RNA chains in isolated nuclei does not generally occur
Results will show transcription rates and an idea of which genes are transcribed

58. Nuclear Run-On Transcription Diagram
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

59. Reporter Gene Transcription
Place a surrogate reporter gene under the control of a specific promoter and measure the accumulation of the product of this reporter gene
The 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 a chemiluminescent compound that emits light

60. Measuring Protein Accumulation in Vivo
Gene activity can be monitored by measuring the accumulation of protein, the ultimate gene product
There are two primary methods of measuring protein accumulation
Immunoblotting / Western blotting (discussed earlier)
Immunoprecipitation
Immunoprecipitation typically uses an antibody that will bind specifically to the protein of interest followed with a secondary antibody complexed to Protein A on resin beads using a low-speed centrifuge

61. 5.8 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

62. Filter Binding
Filter binding is used to measure DNA-protein interaction and 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

63. 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

64. 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

65. Footprinting
Footprinting detects protein-DNA interaction and will show 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

66. 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

67. DMS Footprinting
Starts the same way as DNase footprinting but then methylate with DMS at conditions for 1 methylation per DNA molecule
The protein is then dislodged and treated to remove the methylated purines resulting in apurinic sites which breaks the DNA
The DNA fragments are then electrophoresed and autoradiograped for detection
Dimethylsulfate (DMS) is a methylating agent

68. 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

69. Chromatin Immunoprecipitation (ChIP)
ChIP is a method used to discover whether a given protein is bound to a given gene in chromatin - the DNA-protein complex that is the natural state of the DNAnin a living cell
ChIP uses an antibody to precipitate a particular protein in complex with DNA, and PCR to determine whether the protein binds near a particular gene

70. Chromatin Immunoprecipitation (ChIP)

71. 5.9 Assaying Protein-Protein Interactions
Immunoprecipitation uses an antibody that will bind specifically to the protein of interest and, using a low-speed centrifuge, will ‘pull-down’ any proteins associated with the protein of interest
The yeast-two-hybrid assay is used to demonstrate binding (even transient) between two proteins
The yeast-two-hybrid assay can also be used to fish for unknown proteins that interact with a known protein

72. The Yeast-Two Hybrid Assay

73. 5.10 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

74. 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

75. 5.11 Knockouts and Transgenes
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
Foreign genes, called transgenes, can also be added to an organism, such as a mouse, to create transgenic mice

76. 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 will occur between the altered gene and the resident, intact gene
These events are rare, many cells will need to be screened using the introduced genes

77. Making a Knockout Mouse: Stage 1

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

79. Making a Knockout Mouse: Stage 2

80. 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

81. Methods to Generate Transgenic Mice
Two methods to generate transgenic mice:
1. Injection of cloned foreign gene into the sperm pronucleus just after fertilization of a mouse egg but before the sperm and egg nuclei have fused to allow for insertion of the foreign DNA into the embryonic cell DNA
2. Injection of cloned foreign DNA into mosue embryonic stem cells, creating transgenic ES cells
Both methods produce chimeric mice that must undergo several rounds of breeding and selection to find true transgenic animals

82. Summary
To probe the role of a gene, molecular biologists can perform targeted disruption of the corresponding gene in a mouse and then look for the effects of the mutation in the ‘knockout mouse’ or insert the foreign gene as a transgene in the ‘transgenic mouse’