topic 8 amplifying DNA fragments Flashcards
why do you need to amplify a DNA fragment?
to make a sufficent quantity to work with
what are the two methods to amplify a DNA fragment?
in vivo cloning and in vitro cloning
how does in vivo cloning occur?
DNA fragment is inserted into a vector
vector transfers the DNA fragment into host cells
identify transformed host cells
how is the DNA fragment inserted into a vector?
vector DNA is cut open using the same restriction endonuclease that was used to isolate the DNA fragment containing a target gene
the sticky ends of the vector are complementary to the sticky ends of the DNA fragment containing the gene
vector DNA and DNA fragment are mixed together with DNA ligase
DNA ligase joins the sticky ends of the DNA fragment to the sticky ends of the vector DNA
process is called ligation
new combination of bases is called recombinant DNA
what is a vector?
something that is used to transfer DNA into a cell
plasmids (small, circular molecules of DNA in bacteria) or bacteriophages (viruses that infect bacteria)
how does the vector transfer a DNA fragment into host cells?
vector with the recombinant DNA is used to transfer the gene into host cells
if a plasmid vector is used, host cells have to be persuaded to take in the plasmid vector and its DNA
if a bacteriophage vector is used, the bacteriophage will infect the host bacterium by injecting its DNA into it
the phage DNA integrates into the bacterial DNA
host cells that take up the vectors are transformed
what percentage of host cells take up the vector and its DNA?
around 5%
what are marker genes used for?
identifying the transformed cells
how are transformed host cells identified?
marker genes inserted as vectors at the same time as the gene to be cloned
host cells grown on agar plates
cell divides and replicates its DNA to create a colony of clones
all the cells now contain the cloned gene and marker gene
marker gene can code for antibiotic resistance- host cells
