MCGB - Intro To Molecular Techniques Flashcards
What are endonucleases and what do they do?
Enzymes that recognise and degrade foreign DNA. Specific endonucleases recognise specific DNA sequences. Also known as restriction enzymes - “molecular scissors”.
What is DNA gel electrophoresis?
A method for separation of DNA based on size and charge.
In DNA gel electrophoresis, does the DNA move towards the positive or negative electrode?
Positive (as DNA is negatively charged) - smallest will move furthest
What are the requirements for gel electrophoresis?
- gel (matrix allows separation of fragments)
- buffer (allows charge on DNA samples across gel)
- power supply (generates charge difference)
- stain/detection (to identify DNA)
Give some reasons why restriction analysis might be used.
- investigate size of DNA fragments
- investigate mutations
- investigate DNA variation
- clone DNA
What are plasmids?
Small, circular dsDNA found in bacteria. They can transfer to other bacteria and often carry genes for antibiotic resistance.
What are the four basic steps of gene cloning?
- ISOLATE gene following restriction enzyme digestion
- INSERT gene into plasmid vector
- INTRODUCE recombinant DNA molecule into host cell
- IDENTIFY + ISOLATE the clone containing DNA of interest
Give an example of human gene cloning that produces a useful substance.
Synthesis of pro-insulin by bacteria
What are the steps of PCR?
- dsDNA split (95 degrees)
- primers bind to DNA (60 degrees)
- DNA extended from primers by polymerase (72 degrees)
Denaturation -> annealing -> polymerisation
Give some reasons for using PCR
To amplify a specific DNA fragment, in order to investigate single base mutations/small deletions or insertions/variation, genetic relationships
What is protein gel electrophoresis?
Separation of proteins on basis of size, shape or charge
What are the requirements for protein gel electrophoresis?
Same as those for DNA gel electrophoresis - gel, buffer, power supply, stain/detection
Which way do the protein samples move in protein gel electrophoresis?
Towards positive electrode.
What is SDS-PAGE?
A method of gel electrophoresis where SDS (an anionic detergent) binds to a polypeptide chain in relation to its Mr, allowing categorisation of polypeptides by Mr.
What is IEF (isolectric focusing)?
A tube full of gel has a pH range along its length. Proteins migrate til they reach a pH equal to their pI. This allows separation of proteins by their charge.
What is the difference between 2D-PAGE and SDS-PAGE?
2D-PAGE involves separation of proteins by pH and Mr simultaneously, while SDS-PAGE is just Mr.
How would an entire protein be identified?
- digested with trypsin
- undergo mass spec
- generate list of peptide sizes
- use database to identify protein
What is an epitope?
The part of an antigen molecule to which an antibody attaches itself.
Give some differences between polyclonal antibodies and monoclonal antibodies.
Polyclonal are produced from many B lymphocytes, have many different antibodies and multiple epitopes.
Monoclonal made from 1 B lymphocyte, one identical antibody, one epitope.
How does an enzyme-linked immunoabsorbent assay work?
Antigen attached to surface, specific antibody (with enzyme linked to it) applied over surface allowing it to bind. Enzyme’s substrate is added, which makes it change colour, this is proportional to amount of specific antibody
What is the V0 in an enzyme-based reaction?
Initial rate of reaction
Give two examples of continuous enzyme assays.
Spectrophotometry and chemoluminescence
Give two examples of discontinous enzyme assays.
Radioactivity and chromatography
True or false - enzymes can be used to measure clinically important metabolites?
True, eg test strips or glucose monitor
