GENE TECHNOLOGIES TOPIC II Flashcards
What is an endonuclease?
Another name for restriction enzyme that bacteria use to destroy (chop up) any foreign DNA that comes into the cell from a phage.
Where does the EcoR1 enzyme cut between?
Between the G and A
What do the bacteria use protect their own DNA from being digested by the enzymes and how does this work?
They modify their DNA with methyl groups which are added to A or C bases in major groove. Methyl groups block the binding of restriction enzymes BUT can still be replicated and read.
What are microarrays?
Small genome chips that can be designed for becterial genotyping. Also analyse gene expression by monitoring mRNA products of thousands of genes at once
How are microarrays made?
spotting nucleic acids onto glass slides (preare probes then incubate chips with probes and then attach them covalently)
- Can also generate probes in situ (oligonucleotide probes)
What other things to rerstriciton enzymes (TYPE II) require to cut sequences?
Mg2+ but NOT ATP
- Always cut both strands in the same way
- Most sites are 4-8 nucleotides long and palindromic
What are the major uses for endonucleases?
Generation of reproducible, manageable-sized family of fragments (sequencing)
- Gene cloning (recombinant protein expression like insulin)
- DNA profiling/fingerprinting (comparative studies, forensics)
- Gene mapping
- Gene disruption (genome engineering)
What is a polylinker section in a plasmid?
An area to facilitate cloning
What does an expression vector contain?
A promoter sequence (whereas a cloning vector doesn’t
What is site-directed mutagenesis?
A combination of PCR and cloning; PCR with modified oligonucleotides- this results in change in the protein sequence (mutation, deletion, insertion)
In site-directed mutagenesis, how is template DNA removed?
It is degraded by using Dpn I - methylation dependent restriction endonuclease
What is gene replacement?
Where normal gene is replaced which gives info on activity of mutant gene without normal gene being in the way
What is gene knockout?
Where the normal gene is inactivated completely to see the side effect of losing the normal gene
What is gene addition?
Mutant gene added to see whether it is dominant over normal gene or if it has a recessive effect
What is a conditional mutant?
A gene expressed where you want it and when you want it (temporal and spacial specificity)
