What is a genome?
All genes in an organism/cell
What is the proteome?
All of the proteins that can be made by the genes in an organism/cell
Why is it easier to determine the proteome of simple organisms rather than complex organisms from their genome?
Simple organisms have less non-coding DNA
What must be done to DNA in order to sequence the entire genome?
Must be broke into fragments
What is recombinant DNA technology used for?
To combine genetic material from different sources
What are organisms that contain transferred DNA known as?
Transgenic organisms
What are the three ways that DNA fragments can be produced/isolating the gene?
1 - reverse transcriptase
2 - restriction endonuclease enzymes
3 - gene machine
What are the 5 stages of gene cloning?
1- Isolation of the DNA
2 - Insertion
3 - Transformation
4 - Identification
5 - Cloning/growth
Explain the process of using reverse transcriptase to isolate a gene
- Use a cell that expresses desired protein
- Since cells only contain two copies of each gene, obtaining a DNA fragment is hard so mRNA is obtained instead since many mRNA molecules can be synthesised from a gene.
- Isolate mRNA from the cell and use reverse transcriptase to convert it to cDNA (complementary DNA)
- Use DNA polymerase to produce double-stranded molecule
Explain the process of using restriction endonucleases to isolate a gene
- Restriction enzymes directly cut the gene out
- They cut DNA at specific sites which are called recognition sequences or restriction sites and are palindromic
- Straight ends - cuts occur between two opposite base pairs leaving blunt ends
- Staggered ends - Cuts occur that are not opposite base pairs leaving sticky ends
Explain the process of using a gene machine to isolate a gene
- The sequence for the target gene is obtained from a database.
- Nucleotides are added in the correct order to synthesise the correct base sequence.
- Protecting groups are added throughout the synthesis to make sure the correct nucleotides are added and no side branches are produced.
What are the two different types of gene cloning
- In vivo - within a living organism
- In vitro - outside of a living organism
Explain the process of inserting a gene
- The vector DNA (usually a plasmid) is isolated
- The vector DNA is cut open using the same restriction endonuclease as was used to isolate the gene. This means the sticky ends (if sticky) of the vector DNA are complementary to the sticky ends of the DNA fragment containing the gene
- Ligase is the enzyme that joins the gene fragment and the vector together. This process is known as ligation.
- The new combination of bases in the DNA is called recombinant DNA (rDNA)
Explain the process of transformation
- The vector with the rDNA is used to transfer the genes into cells (host cells)
- The host cell taking up the vector containing the desired gene is said to be transformed
Explain the process of identifying transformed cells
- Only some of the host cells will take up the vector and the desired gene.
- Marker genes can be used to identify cells which have been transformed
- The marker gene can code for antibiotic resistance so host cells are grown on agar plates containing a specific antibiotic. Only transformed cells that have the marker gene will survive and grow.
- Or the marker gene can code for fluorescence so the agar plate is places under a UV light, cells that fluoresce contain religated vectors and cells that contain desired gene will not fluoresce as the gene interrupts the second marker.
- A final way of identification is to add a substrate, if the colour does not change, then the enzyme doesn’t function meaning that the desired gene has been inserted.
What reaction does in vitro cloning use?
The polymerase chain reaction (PCR)
Explain the different stages of PCR
- DNA is heated to 95 degrees to break the hydrogen bonds between the DNA strands
- The mixture is then cooled to 55 degrees and primers and DNA nucleotides are added.
- Primers bind (anneal) to the ends of the strands to stop them re-joining together
- The reaction mixture is heated to 72 degrees so DNA polymerase can work and create new strands of DNA alongside the template strands.
How many new copies of the DNA does one PCR cycle create?
2
Why are primers needed in PCR?
To stop the chains re-joining
Why should the DNA polymerase be heat resistant in PCR?
To withstand the 95 degrees that the mixture is heated to when separating the DNA strands. (So it isn’t denatured during this)
What are the benefits of transformed organisms?
Agriculture:
- Crops can be modified to give higher yields.
- Crops can be transformed to have resistance to pests or droughts.
Industry:
- Can be used to manufacture enzymes used in industry (e.g. cheese-making)
Medicine:
- Can be used to produce drugs or hormones
What are the ethical concerns of transformed organisms?
Agriculture:
- A gene for herbicide resistance could pass on to a weed creating a mutant super weed
- Producing the same crop all the time can reduce biodiversity
Industry:
- People think they may not have a choice whether to eat genetically modified food or not if the food isn’t labelled correctly
Medicine:
- Could lead to unethical uses of genetic engineering (designer babies)
What is gene therapy?
Introducing a healthy gene to alter the effects of a defective gene
How does gene therapy work if a disorder is caused by a mutated dominant allele?
You can ‘silence’ the dominant allele (e.g. by sticking DNA in the middle of the allele to stop it from working)
