Genetic Engineering Flashcards
1
Q
Gene Transfer - Universal Genetic Code
A
- The genetic code is universal
- All DNA nucleotides across species are the same and a codon of bases will code for the same amino acid regardless of the species it is in
- This means that when the gene of one organism is transferred into another organism, that organism will transcribe to produce the same protein (with the same sequence of amino acids)
- An organism that contains the DNA of another organism is said to be a transgenic organism
2
Q
The process of transferring genes - restriction enzymes
A
- DNA is extracted from the donor cell and then the desired gene is removed from the DNA by restriction enzymes
- Restriction enzymes target a specific sequence of bases known as a recognition site. There are two ways that the DNA can be cut
1. Sticky End restriction enzymes
2. Blunt End restriction enzymes
3
Q
The Process of Transferring Genes - Sticky End
A
- Sticky end restriction enzymes: they cut the DNA at its recognition site to produce a DNA fragement with two sticky ends
- A sticky end is an exposed single strand overhang of nucleotide bases. These exposed bases are then able to bind due to complemetnary base pairings to a new DNA strand
4
Q
The Process of Transferring Genes - Blunt Ends
A
Blunt end restriction enzymes cut DNA at its specific recognition site to produce a cut with blunt ends. This means that there are no exposed nucleotide bases
5
Q
The Process of Transferring Genes - Vector
A
- A suitable vector (a way of transferring the desired gene into the new cell) is obtained
- Often phage viruses (a virus that infects bacteria) or plasmids (a circular loop of DNA found in bacteria) are used
- Plasmids are capable of replicating within a cell independently of the main DNA
- The plasmid is then cut using the SAME restriction enzyme used on the desired gene. This will result in the plasmid having sticky ends that are complementary to the gene
6
Q
The Process of Transferring Genes - DNA ligase
A
- The isolated gene is then mixed with the vector together with the enzyme DNA ligase. The gene will insert itself into the plasmid due to the bonding between the complementary base pairs. These are created due to the use of the same restriction enzyme (which cut the gene and plasmid at the same recognition site).
- the DNA ligase will form the sugar-phosphate backbone between the gene and plasmid
- This process is called gene splicing
7
Q
The Process of Transferring Genes - multiplication
A
- The vector is then cloned so that there are many copies available to be taken up by the host cell
- The plasmid is then introduced into the bacteria by placing it in a bacterial culture. This culture is then treated with divalent cations so some of the bacteria will take up the recombinant placid
- The host cell will then express the inserted gene and produce the protein for which it codes
- For organisms that are unicellular, they can be cultured in large vats under suitable conditions to create large numbers of the bacteria. Each bacteria producing the desired protein, which can be collected purified and used
8
Q
Applications of recombinant DNA
A
- The creation of bacteria and yeast able to produce the hormone insulin for use in treating type 1 diabetes
- Transgenic bacteria capable of producing human growth hormone which can be used by children suffering from a deficiency
- Transgenic yeast capable of producing factor VIII protein which is essential for normal blood clotting. This is used by those suffering from haemophilia who need regular injections of factor VIII
9
Q
Recombinant Vaccines
A
- With recombinant vaccines, the gene that is transferred is the one that codes for an antigen of a pathogenic organism
- This gene is cut out of the pathogen’s DNA and the inserted into the vector (a yeast or harmless virus)
- The vector will then display the antigen on its surface so when injected into a human produces an immune response; forming the relevant memory lymphoctyes to the antigen
- The vector being harmless to the person will not make them sick. This provides an advantage over attenuated vaccines which occasionally revert back to their harmful form
10
Q
Recombinant vaccines: Sub-Unit vaccines
A
- The antigens can be removed from the vector and then injected into the person. This is known as a sub-unit vaccine. This type of vaccine is used for hepatitis B and Gardasil
11
Q
Recombinant vaccines - other potential uses
A
- production of hybrid vaccines in which the vector has several genes coding for different antigens inserted into its DNA. This means that the vector would express the antigen of several pathogens; providing one vaccine protecting against several diseases
- Another application would be insertion of the genes into attenuated forms of the bacteria that cause gut infections. This means that these bacteria are able to survive in the digestive system so allow the development of oral vaccines. This means that in less developed countries, they can be administered by untrained people and minimise the risk of infection and needle costs
12
Q
Antibiotic Resistance Marker Genes
A
- used to identify the bacteria that have taken up the foreign (e.g. human) DNA. The plasmid used often carries two genes that provide the resistance to the antibiotics ampicillin and tetracycline. Without this plasmid, the bacteria have no antibiotic resistance genes
- A single restriction enzyme recognition sequence lies within the tetracycline resistance gene. A foreign gene, spliced into this position, will disrupt the tetracycline resistance gene, leaving the bacteria vulnerable to this antibiotic
- It is possible to identify the bacteria that successfully take up the recombinant plasmid by growing the bacteria on media containing ampicillin, and trasnferring colonies with both antibiotics
- Splicing occurs for both the resistance gene and the structural gene in the plasmid, the bacteria that takes up one or the other will have taken up both (as splicing in the plasmid has occured for both). The bacteria that survives in the dish with the antibiotics and nutrient culture will be the ones that have taken up both the structural gene and the resistance gene
