gene cloning
- Created by: vezting
- Created on: 24-02-16 09:06
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- gene cloning
- genetic engineering
- is the manipulation of an organisms DNA. when they have DNA altered they are said to be transformed and their DNA is recombinant
- 1: desired DNA fragment isolated using rest. enzymes
- 2: insert DNA into vector. Eg plasmid or bacteria phages.
- 3: vector DNA is isolated using same rest. enzymes DNA ligase used to stick DNA fragment and vector together
- sticking DNA and vector together
- 1: as vector and DNA have been cut using same rest. enzymes their stick ends are complimentary to each other
- 2: mixed together with DNA ligase, forming sugar phosphate backbone
- 3: new combination of bases- recombinant DNA
- transforming the cells
- heat shock- ice cold calcium chloride solution, cell walls permeable, mixture heated 42 degrees, 1-2 mins.
- electroporation - bacteria given an electric shock, makes it temporarily permeable, allowing plasmid in
- benefits of taking up plasmid- genes that code for resistance are within, contain genes that help microorganism invade host, genes allow micro.to use nutrients they wouldn't normally
- in vivo vs PCR (in vitro)
- in vivo +
- cheaper as bacteria grows on cheap materials
- large fragments can be cloned
- useful if you don't know the exact sequence location of the gene you want to clone
- less technically difficult as PCR needs optimum conditions
- in vivo -
- DNA fragment has to be isolated and this takes a long time
- a lot of space and equipment needed
- in vitro +
- only replicates DNA of interest no isolation needed
- fast process, millions of copies in a few hours
- safer as not dealing with living cells
- uses less space so saves money
- in vitro -
- primer and chemicals expensive
- more mutations as DNA polymerase isn't good at 'proof reading'
- can only replicate small DNA fragment
- in vivo +
- genetic engineering
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