The control of gene expression AQA A2 Biology PART 6 of 6 TOPICS
Gene technologies allow the study of gene function allowing a better understanding of organism function and the new design of industrial and medical processes – recombinant DNA technology:
Recombinant DNA technology involves the transfer of fragments of DNA from one organism or species to another. Since the genetic code is universal, as are transcription and translation mechanisms, the transferred DNA can be translated within cells of the recipient organism. A fragment of DNA would be made if we wanted it in vast quantities. An example would be for the insulin gene. This would be wanted in vast numbers because of diabetics who do not produce enough or do not produce any insulin at all.
Fragments of DNA can be produced by several methods:
- Reverse transcription:
- DNA fragments are produced from mRNA using the enzyme reverse transcriptase. This is done by identifying the cells that make the large amounts of proteins from the desired gene. These cells also contain a large amount of mRNA. Continuing with the example of the insulin gene, the mRNA would be found in the beta cells of the islets of Langerhans in the pancreas.
- The mRNA is then isolated by centrifugation. NB: You do not need to know the step by step process of centrifuging.
- Free DNA nucleotides are then added to the mRNA and the enzyme reverse transcriptase which makes a complementary/copy DNA from the mRNA. NB: Complementary/copy DNA can be abbreviated into cDNA but it is best if you use the full name.
- The mRNA is then broken down by another enzyme leaving just the complementary/copy DNA. NB: The enzyme that is involved in this is called ribonuclease H but this name does not need to be known for the exam.
- As the complementary/copy DNA is single stranded, DNA nucleotides are added and DNA polymerase to make the DNA double stranded with the desired gene so that it can be inserted into the host. The double stranded complementary/copy DNA does not have introns as the mRNA did not have any introns in the first place because the introns are removed leaving the exons to be spliced together.
- Restriction enzymes: NB: The different types of these enzymes do not need to be known as well as the palindromic sequence that they cut into. This will be given to you in the exam in a question where a certain enzyme cuts in between two bases of a specific palindromic sequence.
- These enzymes catalyse a hydrolysis reaction by breaking the phosphodiester bond between nucleotides at certain places. These enzymes make small DNA fragments.
- The enzyme cuts between two bases in a palindromic sequence on both strands of DNA. This makes ‘sticky ends’ (ends which have exposed bases which can bind to complementary bases. This is known by the word anneal). An illustration of this bullet point is shown where it shows what a palindromic is and the process of cutting:
If the restriction enzyme EcoR1 cuts in between G and A in the sequence GAATTC (shown in italics) it creates sticky ends. It recognises the palindromic sequence from 5′ to 3′. Other enzymes also create sticky ends using different palindromic sequence:
The dotty line shows where EcoR1 cuts.
- The same restriction enzyme should be used for the cutting of the DNA of the vector (the host that will carry the desired gene). This is so complementary bases can be made to attach to the sticky ends (shown by the bold and italic bases. DNA ligase then sticks the DNA fragment and the genome of the vector together called ligation:
- Gene machine:
- Technology has been developed so that fragments of DNA can be made from scratch without the need of a DNA template.
- The desired sequence is made.
- The first nucleotide is fixed to a support e.g. a bead.
- Nucleotides are then added one at a time in a process where the protecting groups are added. Protecting groups prevent unwanted branching by making sure that the nucleotides are joined up correctly.
- Short DNA sequences known as oligonucleotides (up to 20 nucleotides long) are obtained. Once these are made the support is removed as well as the protecting groups. The oligonucleotides are then joined together to make a longer DNA fragment.
There are two types of methods as to how the fragments of DNA with the desired gene are amplified. This means how to clone the DNA to obtain high numbers of the desired gene:
- In-vivo amplification:
- This involves using hosts that are cells e.g. bacteria or bacteriophages. Bacteria are always used as they can replicate quickly.
- The DNA fragment is obtained and inserted into the host’s vector outside of the host by the restriction enzyme. If bacterial cells are used the vector would be the plasmid (contains the bacteria’s DNA) or bacteriophages (bacteria that can be infected by viruses).
- The new combination of DNA in the plasmid is called recombinant DNA.
- The vector has to be taken up by the host. If a bacteriophage is used the virus infects the bacteria with the recombinant DNA which is then incorporated into the bacteria’s DNA.
- The cells have to be identified as to which ones have the recombinant DNA and which ones do not. Therefore a genetic marker has to be used. This is an extra gene coding for a special characteristic which is attached to the DNA fragment. So the genetic marker could be coding for a fluorescent protein. Colonies of bacteria can be grown on an agar plate where some of the colonies will glow and some will not under UV light. This shows that the colonies that glow have the recombinant DNA. This is so only the right type of bacteria with the recombinant DNA can be grown in the fermenter in the right conditions. NB: Students will not be required to recall specific marker genes in a written paper.
- In order for the fragment of DNA to be expressed a promoter and terminator sequence must be added or is already part of the DNA to allow this to happen. Without these the desired gene will not be expressed properly.
- In-vitro amplification:
- A mixture is set which has the DNA sample, free nucleotides, primers and DNA polymerase.
- The DNA is heated to 95 degrees to break the hydrogen bonds between the two strands.
- The mixture is then cooled to 55 degrees so the primers can bind (anneal) to the strand. Primers are short DNA molecules that have complementary bases to the DNA of the desired fragment.
- The mixture is then heated to 72 degrees for DNA polymerase to work. The enzyme lines up the free DNA nucleotides up and a new strand is formed from 3′ to 5′.
- The process is repeated again so that more fragments are obtained.
Benefits to humans:
- Agricultural crops can be transformed to create higher yield or are more nutritious. This means that malnutrition and famine is brought under-control and kept at a minimum. Some crops have been genetically modified to withstand pests which keeps the use of pesticides to a minimum making the method cost-effective and is environmentally friendly. It also keeps bioaccumulation at its lowest.
EXAMPLE: The golden rice has a gene from a maize plant and another from soil bacterium which enables beta-carotene to be formed. Beta-carotene allows our bodies to absorb vitamin A and is of particular benefit to people in less developed or developing countries in parts of South-Asia and Africa where vitamin A deficiency is common.
- Biological catalysts are used in industries and can be made from transformed organisms. These enzymes are produced in large quantities for a low price being cost-effective
EXAMPLE: An enzyme is used in industry to make cheese from a transformed organism. Before this method was used an enzyme was used from a cow’s stomach to make cheese. Now, cows are not killed for the enzyme making some types of cheese vegetarian.
- Many different drugs and vaccines are made by transformed organisms using recombinant DNA. They can be made quickly, cheaply and in large quantities.
There are problems with these aspects:
- One type of crop is only grown in the farm which means all the plants are prone to one type of disease.
- There is a possibility of supersedes, weeds which are resistant to herbicides. This would only be the result of crop breeding with wild plants
- Some consumer markets, particularly within the EU, do not import GM foods and produce. This means there is a loss of profit.
- Without proper labelling, customers will not have the choice between GM foods.
- Money may be used for technologies to support genetic modification making less money being used for resources.
- Currently it is illegal to design babies but people are still concerned that the technology will be used unethically.