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Lecture 7: Recombinant DNA techniques - Coggle Diagram
Lecture 7: Recombinant DNA techniques
Restriction Enzymes
background
purified from bacteria
cut at specific sites
bacteria made to attack incoming viral DNA
diff enzyme = diff sequence specificity
Type II - cut at or near restriction site
recognition site=sequence dependent
cut strands in the same way
4-8 nucleotides long
Palindromic
Requires Mg2+ (not ATP)
EcoRI (named after bacteria)
makes sticky ends at GAATTC
will not cleave methylated DNA (at A)
Hpal (named after bacteria)
blunt ends
Uses
make into manageable size fragments
Gene Cloning
DNA Profiling/ Fingerprinting
Gene Mapping
follow the creation / destruction of sites due to mutation of the gene sequences
Gene disruption
Genome engineering using the Crisp-Cas9, Transcription activator-like effector nuclease (TALEN) and Zinc finger nucleases
Cloning DNA Fragments
Steps
Plasmid cut open w restriction enzymes
Insert digested w same enzyme
Insert DNA into plasmid
Mix plasmid and insert - DNA ligase
Recombinant plasmid inserted into competent bacteria
Cells divide, plasmids multiply
Selection of transformed bacteria in presence of antibiotic
Typical E. Coli vector
Inducible expression:
repressor gene
operator sequence
Selection
antibiotic
blue-white selection (LacZ gene disruption)
Efficient expression:
Ribosome-binding site
Transcription termination
sequence
Promoter
Polylinker/ multiple cloning site
Multiple restriction sites to facilitate cloning
Directional Cloning
Use diff restriction sites on each end of DNA
plasmid digested w same enzyme
Otherwise, self ligation could occur
Uses
DNA amplification
Genomic Library
DNA sequencing
Protein expression
Understand gene function - medicine
Transgenic animals and plants
Understand Gene function - biotech
vaccines and therapeutics
enzymes and biomaterials
Gene therapy vectors
Recombinant protein expression
Coding sequence inserted into expression vector
promoter region
Cellular host important
how to insert engineered genes into host cell
into mammalian cells via transfection, microinjection or a virus
into plants via Agrobacterium tumefaciens or particle bombardment
into bacteria via electroporation, heat-shock or chemical transformation
Site directed mutagenesis
Alter specific sequences
PCR with altered oligonucleotides
change in protein sequence
Template DNA is degraded by using methylation-dependent restriction endonuclease (e.g. Dpn I)
changes in gene
Gene Deleted
Gene added on (but recessive against new gene)
Gene Replaced
conditional gene mutation
Regulatory gene mutations
Bacterial DNA binding domain fused to an eukaryotic activator of transcription.
Inhibition of the DNA-binding domain by a small molecule leads to gene inactivation
Tissue-specific gene activation if the activator fusion protein is only expressed in certain tissues.
Understanding protein function
Steps
Synthesize DNA primers for PCR
PCR
determine protein sequence
Gene/ cDNA (Manipulate and introduce altered gene into cells /organism)
Loss of function mutation - delettion
Conditional loss of function mutation - change temp
insert protein coding region of gene into vector
Introduce into E Coli or other host cell to produce protein