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15 Cards in this Set
- Front
- Back
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what are the two different techniques used to make loads of identical copies of a gene |
1)in vitro cloning - where the genes ar nmade outside of a living organism e.g pcr 2) in vivo cloning - where gene copies are made within a living organism - as the organism grows and divides it replicates its DNA |
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what is the first step in invitro cloning |
the DNA fragment is inserted into vector DNA |
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what is a vector |
vectors are used to transfer DNA into a cell - they can be plasmids (small cercular molecules of DNA in bacteria) - or bacteriophages (viruses that infect bacteria) |
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how is the DNA fragment inserted into a vector |
the vector DNA is cut open using the restriction endonuclease - the sticky end of the vector are -complementary to the sticky ends of the DNA fragment -the vector DNA and DNA fragement are mixed together with DNA ligase -dna ligase joins the sticky ends of the DNA fragment to the sticky ends of the vector Dna = ligation |
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what is the vector with recombinant DNA then used for |
it is used to transfer the gene into cels - called host cells |
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if a plasmid vector is used host cells have to be persuaded to take in the plasmid vector, how is this done |
host bacterial cells are placed in ice-cold calcium chloride solutions to make their cell walls more permeable - the plasmids are added and the mixture is heat shocked which encourages the cells to take in the plasmids |
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how does a bacteriophage infect the host bacterium |
by injecting its DNA into it - the phage DNA (with the target gene in it) then intergrates into the bacterial DNA |
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host cells that takeup the vectors containing the genes of interest are said to be what? |
transformed |
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what are marker cells used for |
marker cells can be used to identify the transformed cells |
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how are marker genes used to identify transformed cells |
marker genes can be inserted into vectors along with the gene to be cloned = any transformed host cells will contain the gene to be cloned and the marker gene, -host cells will produce colonies of cloned cells where all the cells contain the cloned gene and the marker gene |
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what is a benefit of identifying transformed cells |
they are allowed to grow more - producing lots and lots of copies of the cloned gene |
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what are the advantages of in vivo cloning |
1) cloning in vivo can produce mRNa and protein aswell as DNA because its done in a living cell ( which has ribosomes and all the enzymes needed to produce them) 2)cloning in vivo can also produce modified DNA, modified mRNA or modified protein - they have modifications added to them e.g sugar or methyl 3)large fragments of DNA can be cloned using in vivo e.g 20-45 kilobases of DNA can be inserted into some plasmids and bacteriophages 4) its cheap |
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what are the disadvantages of of invivo cloning |
the DNA fragment has to be isolated from other cell components, you may not want modified DNA and it can be quite a slow process - because some type of bacteria grow quite slowly |
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what are the benefits of invitro cloning |
it can be used to produce lots of DNA but not mRNA or protein 2) the DNA produced isnt modified - an advantage if you dont want it to be modified 3) this technique only replicates the DNA fragment of interest - this means you dont have to isolate the DNA fragment from host DNA or cell components 4) invitro cloning is a fast process |
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what are the disadvantages of in vitro cloning -PCR |
it can only replicate a small DNA fragment, you may want a modified product, mRNA and protein not made and can be expensive if you want a lot of DNA made |
