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

Applied Molecular Biology