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Applied Molecular Biology and Regulation of Gene Expression Clinical Haematology Cancer Biology Molecular and Medical Parasitology

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Lecture 1: Recombinant DNA (rDNA): -Academic uses -Expression of products useful for treating disease eg. insulin or expression of whole pathways eg. producing antibiotics or morphine -Genetic engineering of microorganisms, animals and plants with improved characteristics -Creation of a synthetic genome There are applications of rDNA in the biotechnology industry: -Pharmaceutical  -Agricultural -Environmental -Waste treatment -Chemical -Food Cloning: -A fragment of the DNA to be cloned is inserted into a cloning vector -The rDNA molecule is introduced into E.coli by transformation -In the E.coli, the vector directs multiple copies of the rDNA to be made -When the host divides, multiple copies of the rDNA are passed to the progeny -A large number of dividing cells containing the same rDNA is a clone -DNA ligase is responsible for joining DNA fragments together Plasmid cloning vectors must have: -An origin of replication -A dominant selectable marker -Unique restriction sites Features in general: -Sequences can be cleaved to have blunt or sticky ends -Sticky ends can be joined back together providing they've been cut with the same restriction endonuclease -Blunt ends cannot be joined back together as easily and so need linker molecules adding to the ends -These allow the ends to be joined together again -Also terminal transferase can be added if the fragment of interest and the vector have been cut with different restriction endonucleases A genomic library: -A collection of clones which together represents the entire genome Frequency: -You can work out the frequency with which a restriction site will be cut -Clarke and Carbon formula -N=ln(1-P)       ln(1-a/b) -For a genomic library from E.coli, size of insert in plasmid vector is 5 kb -For 99% probability of finding a clone of interest 3866 colonies will need to be screened Screening a genomic library for a clone of interest: -Genetic screening -Immunological screening -Nucleic acid hybridisation   Lecture 2
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