Session 7 Flashcards
Describe the theory behind DNA electrophoresis and how this technique can be used to provide information about DNA fragments
Separates DNA fragments on the basis of charge and size
Fluorescent probe is added and visualised under the UV light
Needs a gel matrix, buffer (pH), power supply (generates charge), stain detection usually fluorescent (visualise DNA)
Fragments travel from negative to positive (DNA is negatively charged so repels)
Smaller fragments travel faster
Describe in general terms a number of standard molecular processes such as gene cloning, restriction analysis and DNA sequencing
Gene cloning - identify DNA fragments of interest and use vectors (bacterial plasmids) to carry DNA, introduce vector and DNA into a suitable host, replication occurs, identify and isolate DNA - makes useful proteins (e.g. Insulin), to find out what genes do, genetic screening, gene therapy
Restriction analysis - to investigate size of DNA fragments, mutations, DNA variation, to clone DNA
DNA sequencing - determination of the order in which the nucleotides are bound, uses dideoxynucleotides as chain termination (DNA lacks 3’ OH so not extended by DNA polymerase)
Explain PCR and appreciate its fundamental importance in genetic testing
Repeated cycle of denature (90C), hybridisation, DNA synthesis (75-80C)
Amplifies DNA fragments of interest from one template, very sensitive technique, investigate small base mutations, insertions, deletions, technique of choice for diagnosis of inherited disease, can detect tumours and early stages of infection
Describe the theory between protein electrophoresis and how this technique can be used to provide information about protein structure
SDS-PAGE
Coats the protein, therefore charge does not affect separation (separated on basis of weight)
Proteins are visualised with a dye
Separated on the basis of pI:
Protein migrates through a gel until it reaches a pH that matches the pI
2D-PAGE:
Allows separation of proteins of complex mixtures (same pI, different weights
Proteomics
Understand the basis for the use of enzyme assays
Enzymes are useful clinical markers
Performed at optimum pH, temperature and ionic strength
Appropriate ions and cofactors are added
High substrate concentration leads to optimal enzyme activity
Enzymes in serum usually an indicator of tissue function or damage
Continuous assays - spectrophometry, chemoluminescence
Discontinuous assays - radioactivity, chromatography
Explain how antibodies can be used in immunoassays and Western blotting to detect the presence of proteins
Enzyme-linked immunoabsorbant assay (ELISA):
Antigen coated well –> specific antibody binds to antigen –> enzyme linked antibody binds to specific antibody –> substrate is added and converted into coloured product by enzyme (rate of colour formation is proportional to amount of specific antibody)
Western blotting:
Nitrocellulose replica of gel electrophoregram –> binding of primary antibody –> binding of enzyme linked secondary antibody –> immunoblot
