Molecular Genetics Methods Flashcards
PAGE
Polyacrylamide gel electrophoresis
Two types of gel for nucleic acid analysis
Agarose - for large DNA
polyacrylamide - some small nucleic acids
General steps for nucleic acid analysis
Agarose and polyacrylamide
- Place samples in wells
- Apply potential difference
- Samples migrate based on size and shape
Water overlay
Purpose
Used to prevent oxidising
Issue with single stranded samples
Tend to take up double stranded structure
Denaturing electrophoresis conditions
Maintain a linear conformation of single stranded nucleic acids.
E.g: formamide or urea.
Southern Blotting
Purpose
More definitive determination of the presence of a particular DNA species of interest within your sample.
Better than PCR for larger molecules
Southern Blotting
Method
- Normal gel electrophoresis
- Transfer gel into tank of salt solution.
- Overlay gel with nylon or nitrocellulose membrane
- Incubated with radioactively labeled nucleic acid probe.
- Hybridiation occurs.
- Wash off unbound probes.
Why overlay the gel with nylon or nitrocellulose membrane?
Southern blotting
Causes the salt solution and nucleic acids to be taken up into the membrane.
Northern Blotting
Purpose
Similar to Southern Blotting.
RNA analysed instead of DNA.
Northern Blotting
Method
- RNA extracted
- Samples purified using oligod T dynabeads.
- mRNA is formamide denatured
- mRNA run on urea-polyacrylamide gel for blotting.
- Radioactively labeled probe is designed to be complementary to the mRNA expressed from the gene of interest
Oligod T dynabeads
Northern Blotting
Use PolyT nucleotides to bind to mRNA.
Beads are magnetised so they can be extracted from sample.
Pulse Field Electrophoresis
Purpose
Allows analysis of DNA molecules into the megabase range
Pulse Field Electrophoresis
Method
Like normal blotting, but the direction of the electric field is changed multiple times.
DNA will have to make directional turns and larger DNA will take longer to do this.
Electrophoretic Mobility Shift Assay
EMSA
Purpose
To analyse DNA-protein interactions
Electrophoretic Mobility Shift Assay
EMSA
Method
DNA molecule and protein are synthesised
Subjected to an assay that employs native PAGE conditions.
Run on a non-denaturing gel, bound protein slows down DNA migration
Single strand conformational polymorphism
SSCP Electrophoresis
Purpose
Quick and robust test for the presence of a mutation
Single strand conformational polymorphism
SSCP Electrophoresis
Method
- Uses single stranded DNA - denatured
- Rapid heat-cold transfer
- Uses non-denaturing gels
- Sequence changes will affect shapes and will change the rate.
- Normally PCR is used to amplify DNA
Why rapid heat-cold transfer?
SSCP
Promotes intramolecular interactions and makes single stranded DNA take on a particular structure.
SSCP
Single strand conformational polymorphism
Transient transfections allow…
… short-term examinations to be conducted.
‘Stable’ tranfections enable…
… longer term investigations.
Transient Transfections
Method
- Apply lisosome to cell culture.
- Lisosomal carrier fuses with membrane.
- DNA/RNA in interior released into cytoplasm
- Mix nucleic acid with lipofection reagent
- leave for 20 then add to culture
- leave culture to assay
Why use liposomes?
TRansient transfections
Unilamellar liposomes have a similar structure to the lipid bilayer, so fuse wit hte membrane readily.
Can contain DNA/RNA of interest in an aqueous interior.
Assay
An assay is an investigative (analytic) procedure in laboratory medicine, mining, pharmacology, environmental biology and molecular biology for qualitatively assessing or quantitatively measuring the presence, amount, or functional activity of a target entity.
Stable transfection of cell lines
Method
- Retrovirus-mediated infection.
- Reverse transcriptase copies DNA from the RNA genome.
- Intergrase inserts the DNA copy into host genome.
- Retrovirus binds to cell and inserts its RNA capsid and enzymes into cytoplasm.
- Packaging cells are grown.
- Inserted genome has a marker that tricks virus mechanism into expressing the desired genome.
Why is DNA/RNA integrated into genome?
To protect it from nucleases which scan the cell and degrade foreign DNA.
Transgenic Procedure
- Target vector contains information to be replaced.
- Selection marker is transfected into the cell.
- This can induce homolgous recombination
- This causes mutation in the host chromosome.
Homologous recombination
Causes a swapping around of genetic material in the targeted gene.
Transfection of embryonic stem cells.
- Collected from inner mass of blastocyst-stage embryos
- Transfered to grow in culture.
- Vector is transformed and homologous recombination is induced
- Only cells with resistance marker are replicated.
- These cells are ingected into another embryo which is inserted into a mother.
Purposes of Selection Marker
(2)
Disruption of target gene.
Enabling selection of those cells that have successfully undergone the mutagenic recombination.
Lox P site
Two inverted repeats separated by a spacer (8 nucleotides).
Cre recombinase
Recognises lox P sites and removes the intervening sequences.
Cre expressions can have organ specific promoters, so only work in that organ.
Haemophilia A
Genetic condition caused by the gene that ecodes factor VIII
Factor VIII
A protein required for normal blood clotting
Recombinant
Protein made by genetic engineering
Recombinant factor VIII from hamster cells
Pros
Used because they are a mammalian cell in which stable transfections can be done very efficiently.
Well suited for the recombinant protein production.
Recombinant factor VIII from hamster cells
Cons
Although the factor VIII is identical to that of humans, the post translational modifications are different, so it is attacked by the immune system in humans.
Method for producing factor VIII
7 steps
- Stable transfection of CHO or BHK cells with engineered viral expression vectors for human factor VIII
- Factor VIII production in hamster cells
- Stringent purification of Factor VIII
- Inactivation of any potential contaminating viral particles
- Nano-filtration to remove any viral particle and/or other potential contaminating pathogens present
- Quality control of purified Factor VIII
- Market
You are performing an experiment where you would like to analyse DNA molecules in the size range 500 kilobases-1megabase. Which type of procedure would be most suitable?
A. Standard agarose gel electrophoresis
B. Standard polyacrylamide gel electrophoresis
C. Pulse gel field electrophoresis
C. Pulse field electrophoresis.
Separates depending on size, and can operate above the kilobase range
During SSCP experiments, nucleotide substitutions, such as mutations in single stranded DNAs causes them to migrate at different rates because the nucleotide substitutions…
A. significantly alter the molecular weight of the ssDNA.
B. result in the ssDNA taking on a different shape.
B. They result in the ssDNA taking on a different shape.
You would like to investigate the suspected presence of a point mutation in a patient’s DNA sample and decide to start your investigation with an SSCP experiment. What electrophoresis conditions would you employ during the experiment?
A. Denaturing conditions
B. Non-denaturing conditions
B. Non-denaturing conditions.
Denaturing conditions would prevent the ssDNA from forming secondary structures which would reveal the presence of a point mutation.
During a size determination analysis, which two of the following are you most likely to add formamide to?
A. Single stranded DNA
B. Double stranded DNA
C. Single stranded RNA
D. Double stranded RNA
A. Single stranded DNA
C. Single stranded RNA
Formamide is used to denature single stranded nucleic acids. Double strands are held together by Watson-Crick interactions, so don’t form interactions that can be denatured. Whereas single stranded nucleic acids must be linearised.
During a southern blot procedure, whic of these elements oif the technique enables you to most definitely assess the presence of a DNA species of known size?
A. A size marker ladder electrophoresed alongside your sample.
B. A labeled oligonucleotide
B. A labeled oligonucleotide.
Knowing the size isn’t definitive proof, whereas the oligonucleotide would be specific to the DNA species.
How to tell if something is an expression vector?
Expression vectors habe a promoter (usually CMV) upsteam of a GFP.
You would like to introduce a mutated gene of interest into a mammalian cell line and be able to study the functional effects over the course of several months. Which of these techniques would be most suitable?
A. Heat shock.
B. Lipofection.
C. Retrovirus mediated transfection.
C. Retrovirus mediated transfection
Enables the DNA of interest to integrate into the host genome, maing it stable and protecting it from nucleases.
The aim of your mouse Cre-LoxP engineering project is to “knock-out” a gene of interest specifically in the liver. Which one of these components would be practically relevant to your transgenics workflow?
A. Insertion of LoxP sites flanking the gene of interest specifically in the liver
B. Insertion of a Cre recominase gene into thte genome specifically in the liver.
C. A Cre recombinase gene undre the control of a promoter that is specifically activated in the liver.
C. A Cre recombinase gene undre the control of a promoter that is specifically activated in the liver.
Actually targets the liver as opposed to B, which isn’t specific. A is nice in theory, but must be very difficult to actually achieve, especially without a cre.
