L5: Genetic Recombination & Use Of Recombinase In Cloning Flashcards
Ligation
Joining DNA molecules
Most efficient when DNA ends are held together transiently via cohesive ends
Favoured at low temps
DNA ligase
Join DNA fragments formed during lagging strand synthesis as part of DNA replication
Joining DNA molecules cut with same restriction enzyme
Removal of phosphate from 5’ ends of plasmid prevents self ligation of vector
Genetic recombination
Site specific recombination: DNA chopped in recombinant sites -> rotated 180 degrees ( sequence inverted)
Transposition: transposable element -> transposition
Types of site-specific recombination
Catalysed by recombinase (type of integrase). Recognition sequences for recombinase. Cross over region: where DNA cutting and rejoining occurs
Insertion, deletion & inversion
Cre recombinase
Circularisation recombinase
From phage 1, circularises linear phage genome during infection
4 identical subunits, each has active site Tyrosine
Recognise LoxP site
Two 13 bp inverted repeats flanking 8 bp core sequence
Each monomer binds a 13 bp repeat -> tetramer brings Loxp sites together
Role of tyrosine in Cre recombinase
Forms covalent intermediate with phosphate in DNA
Tyr phosphate conserves energy of cleaved phosphodiester bond -> dna can be rejoined without energy (ATP)
Action of Cre recombinase
Cleaves and rejoins 2 DNA strands at a time
Vector modification using Cre recombinase
Can add or remove drug resistance genes to vector containing LoxP sites
Transfer of foreign gene between vectors
Donor vector cut with restriction enzyme between 2 LoxP sites (add foreign gene) -> DNA ligase -> cre recombinase & DNA between 2 LoxP sites transferred to acceptor vector -> foreign gene now in acceptor (expression) vector
Conditional Gene Targeting
Knocking out specific gene in specific tissue at specific time
Overcomes problem of germline gene inactivation being lethal
Makes use of Cre/LoxP system to excise gene of interest
Conditional Gene Targeting Experimental Approach
Involves making transgenic animals
Initially make 2 types transgenic mice: expressing Cre recombinase with an inducible and tissue specific promoter & with the gene of interest flanked on each side by LoxP
Cross mice to produce offspring with both traits
From inactivation of gene can get info about:
When during development gene is switched on (deleting it will have no effect if it is not turned on)
Whether liver expression of gene is essential for life (absence of gene may only be lethal during embryonic development)
Function of gene in liver (what changes occur in liver when gene is inactivated)
