3.8.4.1. Recombinant DNA Technology Flashcards
What do gene technologies allow us to do?
Allow the study and alteration of gene function = better understanding of organism function + design of new industrial and medical processes.
What is recombinant DNA technology?
Involves the transfer of fragments of
DNA from one organism, or species, to another.
What makes it possible for the transferred DNA to be translated within cells of the transgenic organism?
- Universal genetic code
- Similar transcription and translation mechanisms
What are transgenic organisms?
Organisms which contain transferred DNA (recipient organism)
What does ‘universal genetic code’ mean?
the same DNA base triplets code for the same amino acids in all living things
What does ‘transferred DNA can be translated within cells of the transgenic organism’ mean?
The transferred DNA can be used to produce a protein in the cells of the recipient organism.
Does the recipient and donor have to be from the same species?
No
How can fragments of DNA be produced?
- conversion of mRNA to complementary DNA (cDNA), using reverse transcriptase
- using restriction enzymes to cut a fragment containing the desired gene from DNA
- creating the gene in a ‘gene machine’
Why do we need to obtain DNA fragments?
So that we can transfer a gene from one organism to another. The DNA fragment will contain the gene you’re interested in (target gene).
Using reverse transcriptase (enzyme)
Most cells contain only 2 copies of each gene = hard to obtain DNA fragment which contains the target gene.
BUT contain MANY mRNA molecules (complementary to the gene) = easier to obtain
mRNA templates to make DNA
- RT makes DNA from RNA template. DNA prod is called cDNA
Reverse transcriptase example
Pancreatic cells prod insulin (protein). Lots of mRNA molecules complementary to insulin gene but only 2 copies of the gene itself.
RT used to make cDNA from insulin mRNA
- mRNA isolated from cells
- mix with free DNA nucleotides and RT
- RT uses mRNA as template to synthesise new strand of cDNA
Transcription vs RT
Transcription
DNA —-> mRNA
RT
mRNA —-> cDNA
How do Restriction Endonuclease Enzymes work?
Some sections of DNA = palindromic sequences of nucelotides.
REE recognise specific palindromic sequences and cut (digest) the DNA at these places.
Palindromic sequence of nucleotides
Anti-parallel base pairs (base pairs which are read the same in opposite directions)
E.g GAATTC
CTTAAG
What are specific palindromic sequences known as?
Recognition sequences
Why do different REE cut at different specific recognition sequences?
Because the shape of the recognition sequence is complementary to the enzymes active site.
How are REE used to obtain a DNA fragment?
Recognition sequences present @ either side of DNA fragment, use REE to separate it from rest of DNA
- DNA sample incubated with specific REE
- REE cuts DNA fragment out via hydrolysis reaction
By which reaction do the REE cut the DNA fragment out?
Via a hydrolysis reaction.
Sticky ends
Sometimes cut leaves sticky ends - these can be used to bind (anneal) DNA fragment to another piece of DNA that has sticky ends with complementary sequences
What are sticky ends?
small tails of unpaired bases at each end of the fragment
Using a ‘gene machine’
Technology = fragments of DNA can be synthesised from scratch without need for pre-existing DNA template.
DATABASE containing necessary info to produce the DNA fragment
DNA sequence doesn’t have to exist naturally - any sequence can be made
How does a gene machine work?
- sequence required designed
- first nucleotide in seq fixed to a support e.g. bead
- nucleotides added step by step in correct order, in cycle of processes that includes adding protecting groups
What is the purpose of protecting groups?
Ensures that the nucleotides are joined at the right points, to prevent unwanted branching.
What are the short sections of DNA produced in a gene machine called?
Oligonucleotides (around 20 nucleotides long).
What happens when the oligonucleotides are complete?
Broken off from support and all protecting groups are removed. Oligonucleotides can then be joined together to make longer DNA fragments.
What happens once you’ve isolated your DNA fragment?
It needs to be amplified (make lots of copies of it) so you have a sufficient quantity to work with.
How can fragments of DNA be amplified?
By in vitro and in vivo techniques
In VITRO Amplification
Uses the polymerase chain reaction (PCR)
Copies of the DNA fragment are made OUTSIDE of a living organism using the PCR.
Advantage of PCR
Can be used to make millions of copies of a DNA fragment in just a few hours.
How does PCR work?
Several stages and repeated over and over to make lots of copies.
- Reaction mixture with DNA sample, free nucleotides, primers and DNA polymerase
- Heat mixture to 95 degrees C
- Cool mixture to between 50-65 degrees C
- Heat mixture to 72 degrees C
Two new copies of the fragment are formed and one cycle of PCR is complete.
What are primers?
Short pieces of DNA that are complementary to the bases at the start of the fragment you want
What is DNA polymerase?
Enzyme that creates new DNA strands
Why is the DNA mixture heated to 95 degrees?
To break the H bonds between the two strands of DNA
Why is the DNA mixture then cooled to between 50-65 degrees?
So that the primers can bind (anneal) to the strands.
Why is the DNA mixture then heated to 72 degrees?
So that DNA polymerase can work. It lines up free nucleotides alongside each template strand. Specific base pairing = new complementary strands formed.
What happens once 1 cycle of PCR is complete?
Cycle starts again (mixture heated to 95 degrees).
This time, all 4 strands (two original and two new) are used as templates.
What does each PCR cycle do to the amount of DNA?
Doubles the amount of DNA.
e.g. 1st cycle: 2 x 2 = 4 DNA fragments
2nd cycle: 4 x 2 = 8 fragments
3rd cycle: 8 x 2 = 16 fragments and so on…
In VIVO Amplification
When copies of the DNA fragment are made inside a living organism.
Step 1 of In VIVO
DNA fragment inserted into a vector
- into vector DNA
Vector DNA cut open using same REE that was used to isolate the DNA fragment containing target gene
Vector DNA + DNA fragment mixed together with DNA ligase (enzyme).
What is a vector?
Something that’s used to transfer DNA into a cell - e.g. plasmids or bacteriophages
What is a plasmid?
Small circular molecules of DNA in bacteria
What are bacteriophages?
Viruses that infect bacteria
Why is the vector DNA cut open using the same REE that was used to isolate the DNA fragment?
So that the sticky ends of the vector are complementary to the sticky ends of the DNA fragment containing the gene.
What does DNA ligase do?
Joins the sticky ends of the DNA fragment to the sticky ends of the vector DNA. This process is called ligation.
What is the new combination of bases in the DNA after step 1 of In VIVO amplification called?
Recombinant DNA (vector DNA + DNA fragment)
Step 2 of In VIVO
Vector with recombinant DNA used to transfer gene into cells called host cells.
What happens if a plasmid vector is used?
Host cells have to be persuaded to take in the plasmid vector and its DNA
Example of how host cell is persuaded
Host bacterial cells placed into ice-cold CaCl2 solution to make cells walls more permeable.
Plasmids added and mixture heat shocked (heated to 42 degrees for 1-2mins). This encourages the cells to take in the plasmids.
What happens if a bacteriophage vector is used?
Bacteriophage infects host bacterium by injecting its DNA into it. The phage DNA (with target gene in it) then integrates into the bacterial DNA.
What does transformation of host cells mean?
Host cells that take up the vectors containing the gene of interest are said to be transformed.
Step 3 In VIVO
Only 5% of host cells take up the vector and its DNA = important to identify which cells have been transformed.
What type of gene can be used to identify the transformed cells?
Marker genes - these detect genetically modified (GM) cells or organisms.
How can marker genes be used to identify the transformed cells?
Inserted into vectors at the same time as the gene to be cloned. This means any transformed host cells will contain the gene to be cloned and the marker gene.
- host cells grown on agar plates
- each cell divides and replicates its DNA = colony of cloned cells
When will transformed cells produce colonies?
Where all the cells contain the cloned gene and the marker gene
What can the marker gene code for?
antibiotic resistance
fluorescence
How can the marker gene code for antibiotic resistance?
host cells grown on agar plates containing specific antibiotic, so only transformed cells that have the marker gene will survive and grow
How can the marker gene code for fluorescence?
agar plate placed under UV light, only transformed cells fluoresce
What can identified transformed cells do?
Allowed to grow more, producing lots of copies of the cloned gene.
Addition of promoter and terminator regions to the fragments of DNA
Want the transformed host cells to produce the protein coded for by the DNA fragment.
The vector must contain specific promoter and terminator regions.
What are promoter regions?
DNA sequences that tell RNA polymerase when to start producing mRNA
What would happen without the correct promoter region?
The DNA fragment won’t be transcribed by the host cell and the protein won’t be made.
What are terminator regions?
DNA sequences that tell RNA polymerase when to stop producing mRNA
Where are promoter and terminator regions found?
May be present in the vector DNA or may have to be added in along with the fragment.
What are transformed organisms also known as?
Genetically engineered/ genetically modified (GM) organisms.
What is genetic engineering?
When micro-organisms, plants and animals are transformed using recombinant DNA technology.
How can transformed microorganisms be made?
In VIVO cloning e.g. foreign DNA inserted to produce a useful protein such as insulin
- DNA fragment containing insulin gene isolated
- Inserted into plasmid vector
- Plasmid containing recombinant DNA transferred into a bacterium
- Transformed bacteria identified and grown
- Insulin produced from cloned gene extracted and purified
How can transformed plants be produced?
- Gene coding for desirable protein inserted into plasmid
- Plasmid –> bacterium
- Bacterium = vector to get gene into plant cells
- Correct promoter region added with gene = transformed cells can produce desired protein
How can transformed animals be produced?
- Gene coding for desirable protein inserted into early embryo/ egg cells of female
- very early embryo = all body cells of resulting transformed animal will contain gene
- into egg cell = when female reproduces, all cells of offspring will contain the gene
How can you control which of the animal’s body cells the protein is produced in?
promoter regions which are only activated in specific cell types can control exactly which cells the protein is produced in
What is the advantage of only producing the protein in certain cells?
It can be harvested more easily. Producing it in the wrong cells could damage the organism.
In what 3 ways can recombinant DNA technology and transformed organisms be used to benefit humans?
agriculture
medicine
industry
Agriculture
crops transformed so they give higher yields/ more nutritious
used to reduce risk of famine and malnutrition
transformed to have pest resistance/ so fewer pesticides are needed
reduces costs and reduces env problems associated with pesticides
Example of agriculture
Golden rice = variety of transformed rice
One gene from maize plant and one from a soil bacterium = enable rice to prod beta-carotene (used by our bodies to prod Vit A)
Developed to reduce Vit A deficiency in areas where there’s a shortage of dietary Vit A e.g. SAsia, Af
- up to 500,000 children per year worldwide go blind due to this deficiency
What types of concerns are there about the use of recombinant DNA technology?
ethical
financial
social
Concerns with agriculture
- monoculture = whole crop vulnerable to the same disease (the plants are genetically identical)
- environmentalists say monocultures reduce biodiversity - damages env
- superweeds which could occur if transformed crops interbreed with wild plants
- this could lead to an uncontrolled spread of recombinant DNA (unknown consequences)
- organic farmers crops contaminated by wind blown seeds from nearby GM crops
- can’t sell it as ‘organic’ = lose income
What is monoculture?
When farmers plant only one type of transformed crop.
What are superweeds?
Weeds that are resistant to herbicides.
Industry
Biological catalysts (enzymes) - can be prod from transformed organisms (large quantities, less money = reduces costs)
Example of industry
Chymosin (aka rennin) enzyme used in cheese making. Used to be made from rennet (found in stomach of cows) but now from transformed organsisms.
Large quantities, relatively cheap, doesn’t kill any cows = cheese suitable for veg
Concerns with industry
Anti globalisation activists
- few large biotechnology companies control some forms of genetic E
- use of tech increases = companies get more power = smaller companies out of business bc it’s harder to compete
- No proper labelling = wb consumer choice on whether to consume food made using GE organisms
- Some consumer markets e.g EU won’t import GM foods and products = eco loss to producers who have traditionally sold to those markets
What are anti globalisation activists?
Oppose globalisation (growth of large multinational companies at the expense of smaller ones)
Medicine
Drugs + vaccines prod by transformed organisms using recombinant DNA tech
Made quickly, cheaply + large quantities
Medicine example
Insulin used to treat Type 1 diabetes
used to come from animals
Wasn’t human insulin
Human insulin now made from transformed microorganisms using cloned human insulin gene
What animals did insulin used to come from?
cow, horse or pig pancreases
Why was it a problem that the insulin wasn’t human insulin?
It didn’t work as well.
Concerns with medicine
Companies who own GE tech may limit the use of tech that could be saving lives
Tech could be used unethically e.g. designer babies - but this is currently illegal
What are designer babies?
Babies that have characteristics chosen by their parents
Ownership issues of recombinant DNA tech
Who owns genetic material from humans once removed from body - donor or researcher?
- Individ holds rights to their own genetic info
- Value created by researcher who uses it to develop medicine/ in diagnosis
Some large corporations own patents to
