Lab 5 + 6 Flashcards
Discuss the efficiency of recombinant techniques (e.g., restriction enzymes).
- It is not 100%, and may lead to plasmids with 1 insert, 2 inserts, or none at all.
How is agarose gel electrophoresis used for the separation, identification and purification of DNA fragments?
- DNA has a negative charge and will migrate toward the positive electrode (anode).
- When an electric current is applied to the gel, DNA fragments move through the gel at a rate proportional to the applied voltage.
- The rate of migration is also affected by the size and shape of the DNA.
- Large molecules take longer to “worm” their way through the gel than smaller molecules. More compact molecules, such as supercoiled DNA, can migrate more rapidly than less compact molecules, such as linear DNA, of the same molecular weight.
- This procedure results in the separation of DNA molecules by size and/or shape, with the smaller fragments being closer to the positive electrode and farthest from the wells where the samples are loaded.
How are DNA fragments visualized in gel electrophoresis?
- The DNA fragments can be visualized by the addition of ethidium bromide or a safer alternative is a dye called Sybersafe.
- Sybersafe (or ethidium bromide) is a dye that will intercalate (insert) between the bases of the DNA.
- When the gel is illuminated with ultra violet light, the dyes fluoresce a red/orange.
- It is possible to estimate the size of the DNA fragments by the comparison of the DNA sample of interest to molecular weight markers composed of pieces of DNA of known size.
- Molecular weight markers include several fragments of varying size and are run in a lane adjacent to the sample of interest.
- A photograph is commonly taken to provide a record of agarose gels.
What are restriction endonucleases?
- Enzymes that recognize and cleave double-stranded DNA at specific nucleotide sequences (generally 4-6 nucleotides long).
- For instance, the restriction endonuclease, EcoR1 isolated from Escherichia coli has the following recognition sequence and will cut the DNA between the G and A on each strand.
- This results in a staggered cut (image if the cut pieces were pulled apart, there would be overhanging ends that are often called “sticky ends”).
What does genetic engineering refer to?
- Genetic engineering refers to any changes in genetic makeup that result from the direct manipulation of DNA using various technical methods.
What does genetic engineering involve? [3]
- Often this can involve (1) the addition of a gene to the cell’s genetic makeup, (2) the deletion of a gene, or (3) the mutation of a gene.
Describe how the process of genetic engineering has its starts in microbiology. [3]
- Herbert Boyer identified Restriction Endonucleases (RE) - enzymes that recognize specific sequences in the DNA (a specific order of A, C, G, T) and cut into DNA
- Paul Berg was the first to join DNA from two different sources together.
- Stanley Cohen worked out a way to get bacteria to take up plasmids (and recombinant plasmids).
What are plasmids?
Small, circular double stranded DNA structures that replicate independently of the chromosome.
There are several useful plasmids that have been constructed for genetic engineering (e.g., pUC19). They can be isolated from bacteria, modified, and re-introduced. How does a scientist know which bacterial cells have received a plasmid?
There is usually a selectable marker (or trait) that allows the scientist to know which bacterial cells received a plasmid.
Where has genetic engineering of plasmids been useful?
- Making recombinant human proteins that are used in the therapy of medical conditions.
- In genetic engineering, we can cut into a plasmid, insert a gene that we want the bacteria to transcribe, stitch the plasmid back together, and put it back into a bacterial cell.
- The bacterum transcribes the DNA and the mRNA is translated.
- The bacterium then makes the protein.
What are restriction endonucleases?
RE are naturally occuring enzymes that recognize specific sequences in the DNA (a specific order and number of A, C, G, and Ts) and cut into the DNA.
Where are restriction endonucleases found?
In bacterial cells and archaeal cells
RE may represent a primitive immune system against infection with bacterial viruses (by cutting foreign DNA).
Which RE did Herbert Boyer discover?
One in E. coli; he called it EcoR1.
Both strands of DNA are cut - which linearizes a circular plasmid.
Restriction endonucleases are sensitive and need proper conditions for effective cutting.
Describe these conditions [5].
- Need to be kept cold until ready to be used
- Buffers at correct pH and salts at correct concentrations
- Temperature in which they function e.g., 37 degrees C
- Time to cut
- Use appropriate concentration of enzyme (expressed in enzyme units)
What is an enzyme unit?
1 unit = amount of enzyme required to digest 1 µg DNA in 50 µl of reaction volume in 60 minutes
Cloning plasmids used in recombinant DNA often have one copy of many different restriction endonuclease sites.
True or False?
True.
Describe how Paul Berg’s work helps us make recombinant DNA.
- Paul Berg was the first to join DNA from two different sources together - the two DNAs would have been cut with the same restriction endonuclease.
- DNA from SV40 (a tumour virus that infects monkeys) was joined with DNA from a bacteriophage.
- DNA ligase was used to ‘stitch’ the two pieces together
Describe how Stanley Cohen’s work helped introduce recombinant DNA into a cell.
- Cohen had developed a method to get bacteria to take up plasmid DNA.
- Cells are incubated with DNA in a calcium chloride solution on ice.
- A heat-shock results in DNA crossing the membrane.
Describe Boyer and Cohen’s collaboration.
- Their projects worked out the basics of recombinant DNA.
- They demonstrated that a piece of foreign DNA can be introduced into bacteria, and it can be expressed by the bacteria (i.e., transcribe the DNA and translate the mRNA).
- The manipulated DNA is then introduced into the cell - this can be challenging depending on whether this is a bacterium, animal cell, or plant cell.
Describe enumeration of bacteria by direct microscope count.
- Use a known volume of baterial cells on slide called a counting chamber
- Under the microscope - count cells in several fields of view.
- Average the counts and calculate per volume used
Can’t know what % of cells is alive and what % is dead; good if you need to know how many bacteria are present, and quickly.
Describe bacterial enumeration by M.P.N.
- Inoculate a set of tubes with sample.
- Determine number of tubes with growth
- Use M.P.N. index to estimate counts
- This method requires lots of work, time, space, media, resources, and increases chance of error due to lots of pipetting required.
- Also, not all bacteria can grow in all bacteria, so the MPN method does not actually enumerate all bacteria, just those able to grow in the selected media.
Describe enumeration of bacteria by standard plate count.
- Spread and pour methods
- Bacteria spread throughout a solid surface (agar)
- Grow to form colonies
- 1 single cell yields 1 visible colony (assumption)
- Measures living cells only (dead cells don’t grow!)
- Usually need to dilute cell culture first
- Only enumerating bacteria that can grow in the selected media.
Describe bacterial enumeration by optical density.
- The presence of suspended particles in water
- The degree to which a liquid loses transparency
- This can be masured by the reduction in the amount of light that passes through. This is because the light is being scattered by the sample.
- Higher degree of light scattering, higher turbidity (more particles per volume)
- However, some bacteria/pigment in the cell may absorb light, and not scatter it, which complicates matters.
What is optical density?
- Bacteria cells will scatter the light. The amount of light scattered is proportional to cell concentration.
- Higher optical density (O.D. units) value = higher cell concentration
- The spectrophotometer reports the value as Absorbance
- By convention, we include the wavelength of light used to take the measurement, e.g., O.D.600nm
How can an optical density measurement be used for future experiments?
- Using plate counts and optical density - create a standard curve
- O.D. units vs. cell concentration (cells/mL); both values are units of concentration
Sometimes when we construct standard curves, we make the following observations:
- The light that is deflected by one cell may be deflected back into the path of the detector
- The curve flattens because of the ‘billiard ball effect’ when cell concentration becomes very dense.
What is gel electrophoresis?
The separation of molecules in an electric field.
What does it mean if you’re using a 0.7% agarose gel?
- It means there is 0.7g of agarose in 100 mL of buffer
Describe how an isolated bacterial plasmid is prepped for gel electrophoresis.
- 15 µl of your plasmid is mixed with 3 µl of loading buffer (blue).
- The loading buffer contains bromophenol blue, xylene cyanol and glycerol.
- Bromophenol blue migrates through the gel at the same rate as 400 bp DNA fragment
- Xylene cyanol migrates as 4000 bp fragment
- These dyes help us see how the gel is running without disturbing it.
- Glycerol makes sample “heavy” so that it “sinks” into the wells.
Describe what happens after DNA is loaded into an agarose gel and an electrical current is applied across the gel.
- DNA (-ve charge) migrates towards the +ve end.
- Different sizes and structures (conformations) of DNA result in different migration patterns.
- The gel boxes are designed with safety in mind - you will not be able to touch the gel or buffer while the electric field is applied.
How is DNA in agarose gel visualized?
- Fluorescent dye added before pouring
- Two dyes routinely used: Ethidium bromide (EtBr) and SYBRSafe.
- EtBr intercalates between the bases of DNA - this makes it a potential carcinogen
- SYBRSafe is a safer alternative to use, but it should still be treated as hazardous (wear gloves).
- The dyes will bind to the DNA and fluoresce
- These are called ‘bands’.
- Bands are DNA copies of the same size that migrate together.
What is the purpose of a DNA ladder?
- Run a linear DNA ladder or standard for known sizes of DNA to compare the DNA samples to.
Describe how a DNA ladder can be used to make a standard curve.
- Plot on semi-log graph
- Measure the distance in mm from the bottom of the well to the middle of the band
- Use the distance migrated to discern the size of the DNA.
