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the process of altering genetic material by the purposeful manipulation of DNA
Genetic engineering
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Restriction enzymes (REs) cut DNA at specific 4 – 8 base pair(bp) sequences called these which are usually palindromes
restriction sites
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The DNA fragments produced from restriction enzymes are called
restriction fragments
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REs exist naturally in bacteria,protecting them from what?
invading viral DNA
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these are the type of restriction enzymes normally used in genetic engineering
Restriction endonucleases
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what do bacteria do to their DNA, protecting it from REs?
methylate it
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Production of sticky ends by REs allows what to happen?
the insertion of a foreign piece of DNA, as long as it has the same(complementary) sticky ends
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the production of multiple copies of a gene of interest
Gene cloning
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this is an agent used to transfer DNA
A vector
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what are the 2 most commonly used vectors?
- bacterial plasmids
- viruses
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how is cDNA made?
- by isolating mRNA molecules from the cell
- using reverse transcriptase to make a complementary DNA molecule (cDNA)
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what is the advantage of using cDNA for cloning?
it lacks introns, making it easier for a bacterium to express the correct protein
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discuss gene cloning.
- isolate DNA from 2 sources
- cut both with same RE
- mix the DNA; they will join together because of the base pairing
- add DNA ligase to bond covalently
- put plasmid into bacteria via transformation
- identify cells with the recombinant DNA by their ability to grow in the presence of ampicillin but tetracyline
- clone selected bacteria
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this is a large plasmid that contains just the genes necessary for replication (can carry an insert of 100-300 kb)
The BAC (bacterial artificial chromosome)
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To find the clone, you must create and screen this
a genomic “library” of clones
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You screen the library by creating a radioactive RNA probe that will bind to the gene you are interested in- what is this called?
nucleic acid hybridization
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discuss how one would screen a genomic library.
- transfer cells to filter
- treat cells on the filter to denature them
- add radioactive probe to filter
- expose the filter paper to photographic film
- the colonies containing your gene will expose the film because they are radioactive
- go back to the original plate, scoop out the colony you are interested in, and grow it in mass quantity
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screening library using multiwell plates
- The contents of each multiwell plate is transferred to a nylon membrane
- it is then incubated in a plastic bag containing the radioactive probe
- nylon membrane is then allowed to expose x-ray film
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this contains a highly active bacterial promoter just upstream from the restriction site where you insert the eukaryotic gene
expression vector
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