8 Gene expression: 21 Recombinant DNA Technology Flashcards
What does recombinant DNA technology involve?
The transfer of fragments of DNA from one organism, or species, to another.
Why can tranferred DNA be translated within cells of the recipient organism?
The genetic code is universal, as are transcription and translation mechanisms.
List the different methods to produce fragments of DNA.
- conversion of mRNA to complementary DNA, using reverse transcriptase
- using restriction enzymes to cut a fragment containing the desired gene from DNA
- creating the gene in a ‘gene machine’
What are organisms that contain transferred DNA called?
Transgenic organisms.
Decribe the process of using reverse transcriptase to produce DNA fragments.
- mRNA is isolated from cells.
- The mRNA is mixed wih free DNA nucleotides and reverse transcriptase.
- The reverse transcriptase used the mRNA as a template to synthesise a new strand of complementary DNA.
Describe the process of using restriction enzymes to produce DNA fragments.
- Restriction endonucleases cut DNA at specific recognition sites.
- The DNA sample is incubated with specific restriction endonucleases to cut the desired DNA out.
- Sticky ends are produced which anneal to other DNA fragments with sticky ends by complementary base pairing.
Describe the process of using a ‘gene machine’ to produce DNA fragments.
- The required sequence is designed or taken from a database.
- The first nucleotide is fixed to some sort of support (e.g. a bead).
- Nucleotides are added step by step.
- Protecting groups are added as well to prevent unwanted branching.
- Oligonucleotides (short sections of DNA) are produced.
- The oligonucleotides are broken off the support and the protecting groups are removed. They are joined together to make longer DNA fragments.
Describe the process of amplifying DNA fragments by in vivo techniques.
- The DNA fragment is isolated using restriction endonuclease and inserted into vector DNA.
- The vector DNA is cut using the same restriction endonuclease so the sticky ends are complementary to eachother.
- The vector DNA and DNA fragment are mixed with DNA ligase so the sticky ends join, producing recombinant DNA.
- Host cells take up the vectors with recombinant DNA.
- Marker genes can be inserted with the desired gene.
- Promotor and terminator regions are added along with the DNA fragment so the transformed host cells can produce the protein.
What is a vector?
Something that’s used to transfer DNA into a cell.
How are marker genes used in in vivo amplification of DNA fragments?
What can the different marker genes code for and how can you distinguish
- Marker genes are inserted into vectors as well as the desired gene.
- Host cells are grown on agar plates and transformed cells will contain the desired gene and marker gene.
- The marker gene could code for antibiotic resistance so host cells grown with the specific antibiotic will die so only transformed cells will survive.
- The marker gene could also code for fluorescence so only transformed cells will fluoresce under UV light.
Describe the process of amplifying DNA fragments using in vitro techniques.
Polymerase chain reaction.
- A reaction mixture is set up containing the DNA sample, free nucleotides, primers, and DNA polymerase.
- The DNA mixture is heated to 95C to break the hydrogen bonds between the two strands of DNA.
- The mixture is then cooled to 50-65C so that the primers anneal to the DNA strands.
- The reaction mixture is heated to 72C so DNA polymerase can work.
- The DNA polymerase lines up free DNA nucleotides alongside each template strand, by complementary base pairing, and joins adjacent nucleotides.
- Two new copies of the DNA sample are formed, completing one cycle of PCR.
- The cycle starts again, with all four strands being used as template strands.
- Each PCR cycle doubles the amount of DNA.
What are primers?
Short pieces of DNA that are complementary to the start of the desired DNA fragment.
What are the benefits and concerns of using recombinant DNA technology in agriculture?
+ crops can be transformed so they give higher yields or are more nutritious
+ reduces the risk of famine and malnutrition
+ pest resistance so fewer pesticides needed, reducing costs and environmental problems associated with pesticides
- monoculture could happen, making the whole crop vulnerable to the same disease and reducing biodiversity
- herbicide resistant weeds could grow if transformed crops interbreed with wild plants, leading to uncontrolled spread of recombinant DNA, with unknown consequences
- organic crops can be contaminated by wind-blown seeds from GM crops so farmers can’t sell their crop as organic and lose income.
What are the benefits and concerns of using recombinant DNA technology in industry?
+ enzymes used in industrial processes can be produced from transformed organisms, so large quantities are produced for less money, reducing costs
- as the use of this technology increases, big companies become more powerful, forcing smaller companies out of business
- without proper labelling, people may think they don’t have a choice about whether to consume food made using GM organisms
- some consumer markets won’t import GM foods and products, causing an economic loss to producers.
What are the benefits and concerns of using recombinant DNA technology in medicine?
+ many drugs and vaccines can be made quickly, cheaply, and in large quantities using recombinant DNA technology
- companies who own genetic engineering technologies may limit the use of technologies that could be saving lives.
- people may worry that the technology could be used unethically (e.g. designer babies)
