7.3- gene technology Flashcards
what is recombinant DNA?
what is gene technology?
-manipulation of genes in living organisms
-genes from one organisms may be inserted into another
-may be done within same species (gene therapy) or genes may be transferred from one species to another
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5 stages involved in genetic engineering
1.isolating required gene e.g. insulin
2.inserting gene in a ‘vector’
3.transformation- gene delivered into required cell for protein growth
4.identification of host cells that have taken up the gene
5.grow cells with new gene on a large scale
3 ways to isolate the gene of interest
1.using reverse transcriptase
2.using restriction endonucleases
3.the gene machine
why is detergent sometimes used to isolate DNA?
-breaks down cell membranes
-protein may then need to be removed using digestive enzymes
isolating method 1- reverse transcriptase
-enzyme used is reverse transcriptase
-RNA taken from a cell that produces required protein
-reverse transcriptase found in retroviruses like HIV
-it catalyses a reaction in which complementary DNA (cDNA) is made from mRNA + DNA nucleotides
-result is single strand of cDNA
-DNA polymerase and free nucleotides are used to produce a double strand of cDNA.
isolating method 2- restriction endonuclease
-enzyme is restriction endonuclease
-gene can be removed from chromosome using restriction e
-different restriction enzyme cut DNA at different base sequences
-this is called a recognition sequence
-restriction enzymes are made by bacteria
-They are used to destroy the DNA of bacteriophages
Whys does the enzyme cut the DNA twice?
-The enzyme cuts the DNA backbone twice, therefore, the site “reads” the same way backwards as forwards–a palindrome.
e.g. Hannah
isolating method 3- gene machine
-in lab using computer technology
-scientists examine amino acid sequences in primary structure of desired protein then work backwards to work out the mRNA sequence required to produce this, and then DNA sequence
ligation
-isolated gene is inserted into a vector
-the vector is a piece of DNA that can take the gene into the chosen organism
e.g. plasmid is a vector
how gene is inserted into vector
-same restriction enzymes used to cut out the gene is used to cut open the plasmid
-the broken plasmid has sticky ends that are complimentary to the donor gene
-the donor gene will easily combine with the complimentary sticky ends of the plasmid
-ligase catalyses the ligation reaction that joins 2 backbones of the DNA together
-new DNA is recombinant DNA
transformation
-host cells that take up the vector are said to be transformed
-one way of doing this is by placing host cell in ice cold calcium chloride solution to make cell walls more permeable
-plasmids are then added and mixture is heat shocked (42 for 2 mins) which encourages cells to take up plasmids
methods of transformation-gene guns
-DNA shot into cell at high speed carried on minute golf or tungsten pallets
-some cells survive this and accept DNA as part of their genetic material
methods of transformation-lisosome wrapping
-gene to be inserted is wrapped in lisosomes from lipid bilayer
-these fuse with cell membrane and can pass through to deliver DNA to cytoplasm
methods of transformation-microinjection
-DNA injected into cell through very fine micropipete
-this is manipulated using micromanipulator
-hit or miss so many have to be injected before one takes up DNA successfully
-only 5% will be transformed, marker genes can be used to identify them
