the process of altering genetic material by the purposeful manipulation of DNA
Restriction enzymes (REs) cut DNA at specific 4 – 8 base pair(bp) sequences called these which are usually palindromes
The DNA fragments produced from restriction enzymes are called
REs exist naturally in bacteria,protecting them from what?
invading viral DNA
these are the type of restriction enzymes normally used in genetic engineering
what do bacteria do to their DNA, protecting it from REs?
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
the production of multiple copies of a gene of interest
this is an agent used to transfer DNA
what are the 2 most commonly used vectors?
- bacterial plasmids
how is cDNA made?
- by isolating mRNA molecules from the cell
- using reverse transcriptase to make a complementary DNA molecule (cDNA)
what is the advantage of using cDNA for cloning?
it lacks introns, making it easier for a bacterium to express the correct protein
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
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)
To find the clone, you must create and screen this
a genomic “library” of clones
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
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
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
this contains a highly active bacterial promoter just upstream from the restriction site where you insert the eukaryotic gene