Módulo 1 de 4 · Texto
Applied Molecular Biology
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