Introduction To Molecular Techniques Flashcards
What is the role of endonucleases?
Cuts at phosphodiester bonds and breaks DNA molecules
E.g. restriction enzymes
What are restriction enzymes responsible for?
Recognition and degradation of foreign DNA
Recognise and cut specific DNA sequences (restriction sites)
Mostly palindromes of 4, 5, 6, 8 bp
What are sticky ends and can they be rejoined?
Ends of the DNA molecules where they have been cut, not evenly cut
Hydrogen bonds can be formed between them when they come together again but phosphodiester bonds cannot be
How does DNA gel electrophoresis move DNA molecules?
DNA is negatively charged and will move towards the anode when placed in an electric field
Fragments separated by size, smaller molecules travel faster through gel
What are the 4 requirements for gel electrophoresis?
Gel
Buffer
Power supply
Stain/detection
Why is restriction analysis used?
To investigate size of DNA fragments (e.g small deletions)
To investigate mutations (e.g sickle cell disease)
To investigate DNA variation (e.g. DNA fingerprinting)
To clone DNA
What are the 4 steps of gene cloning?
- Isolate relevant gene of interest following digestion with restriction enzymes
- Insert gene of interest into plasmid vector (recombinant DNA molecule)
- Introduce recombinant DNA molecule into suitable host cell (e.g. E. Coli)
- Identify and isolate the clone containing DNA of interest
Why would you clone human genes?
To make useful proteins e.g. insulin
To find out what genes do e.g. HTT
Genetic screening e.g. Huntington’s, BRCA1/2
Gene therapy e.g. Cystic fibrosis
What happens in PCR?
- DNA heated to 95 degrees, DNA denatures, Hydrogen bonds break
- Primers bind (anneal) to both strands of DNA (define by of DNA wanting to be amplified)
- Thermos table DNA polymerase (Taq) amplifies primers and DNA copied
Why is PCR used?
To amplify a specific DNA fragment
To investigate single base mutations
To investigate small deletions or insertions
To investigate variation, genetic relationships
How does protein gel electrophoresis work?
Proteins are separated on the basis of size, shape or charge
Proteins are charged molecules and will move towards the anode or cathode
What are the requirements for gel electrophoresis?
Gel
Buffer
Power supply
Stain/detection
How does SDS-PAGE work?
Stops all hydrophobic interactions
Gives molecules negative charge in proportion to size so proteins then separated on basis of size
How are proteins identified using proteomics?
Digest protein with trypsin
Perform mass spectrometry
Generate list of peptide sizes
Use database of predicted peptide sizes for known proteins to identify protein
What are the features of polyclonal antibodies?
Produced by many B lymphocytes
Multiple different antibodies
Specific to 1 antigen
Multiple epitopes
What are the features of monoclonal antibodies?
Produced from 1 B lymphocyte
1 identical antibody
Specific 1 antigen
1 epitope
Describe the process of western blotting to detect proteins
Banding pattern is transferred onto nitrocellulose replica of gel electrophoretogram
Binding of primary antibody against protein of interest
Binding of enzyme-linked secondary antibody against primary antibody
Immunoblot forms
Describe the process of enzyme-linked immunoabsorbent assay (ELISA)
Antigen coated well
Wash
Specific antibody binds to antigen
Wash
Enzyme-linked antibody binds to specific antibody
Wash
Substrate is added and converted by enzyme into coloured product
Rate of colour formation is proportional to amount of specific antibody
What are the 2 types of enzyme assays and how can they be measured?
Continuous assays (spectrophotometry, chemoluminescence) Discontinuous assay (radioactivity, chromatography)
