in vivo cloning Flashcards
what are the stages of DNA technology in invivo cloning ?
- create DNA fragments for the gene of interest
- insert DNA fragment into a vector
- transform a host cell with the vector
- identify transformed cells
- grow the host cells by cloning
what are restriction endonucleases ?
- enzymes that are used to cut out the gene (DNA fragment ) of interest
- these enzymes cut out recognition sites leaving sticky ends
what must be done to DNA fragments ?
- must be modified to ensure transcription of these genes can occur
how are DNA fragments modified ?
- promoter regions are added at the start of the DNA fragment
- a terminator region is added at the end of the gene
what is meant by a promoter region ?
-a sequence of DNA which is the binding site for the RNA polymerase to enable transcription to occur
what is a terminator region ?
- it causes RNA polymerase to detach and stop transcription
- so only one gene at a time is copied into mRNA
what is a vector ?
- something to carry the isolated DNA fragment into the host cell
what are the most common vectors ?
- plasmids
what are plasmids ?
- circular DNA separate from the main bacterial genome which only contains a few genes
why are vectors used ?
- because we cannot inject the DNA directly into the cell
how is the DNA inserted into the vector ?
- the plasmid is cut open using the same restriction endonuclease
- this creates the same sticky ends
what does this production of sticky ends on the plasmid allow for ?
- it means that the DNA fragment sticky ends are complementary to the sticky ends on the plasmid
what happens to the DNA fragment and cut plasmid ?
- they are combined
- the enzyme ligase sticks them together
what is it called when the enzyme ligase sticks the dna and plasmid together ?
- anneals them
what is the function of ligase ?
- ligase catalyses the condensation reaction to form phosphodiester bonds between nucleotides
what is the step called to get the plasmid into the host cell ?
- transformation
what must happen during transformation ?
- the cell membrane ofthe host cell must be more permeable
how is the cell membrane permeabillity increased ?
- the host cells are mixed with Ca 2+ and heat shocked (sudden increase in temp)
what does the heat shock enable ?
- it enables the vector to enter the host cells cytoplasm
what will happen when the vector is inserted into the host cell ?
- the gene will be expressed to create the protein required
