1. Chapter 5 Molecular Tools for Studying Genes and Gene Activity

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. 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

7. 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 (isoelectric focusing)

9. 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

10. 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

11. 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

12. 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

13. 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

14. 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

15. 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

16. 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

17. 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

18. 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

19. 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

20. Detecting Nucleic Acids With a Nonradioactive Probe

21. 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

22. 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

23. 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

24. 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

25. 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)

26. 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

27. 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

28. Sanger Method of DNA Sequencing

29. 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

30. 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

31. 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

32. Southern Blots and Restriction Mapping

33. 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

34. PCR-based Site-Directed Mutagenesis

35. 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

36. 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

37. 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

39. 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 (IP)
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

40. 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

41. 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

42. 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

43. 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

44. 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

45. 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

46. 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

47. 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

48. Chromatin Immunoprecipitation (ChIP)

49. 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

50. The Yeast-Two Hybrid Assay

51. 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

52. Making a Knockout Mouse: Stage 1

53. 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

54. Making a Knockout Mouse: Stage 2

55. 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

56. 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