Chapter 14 - DNA Applications In Society Flashcards
Plasmids
Define
Used?
Features?
Circular DNA molecules that replicate more quickly than the bacterial chromosome.
Plasmids are the vectors often used to transport foreign DNA into bacterial cells.
Features:
• a selectable market gene - often a gene that has the allele for resistance to an antibiotic, such as ampicillin. Lack the gene = killed by the antibiotic.
• recognition sites for several restriction enzymes that produce sticky ends, where it is possible to insert foreign DNA.
• lacZ segment
Define recombinant plasmid
Plasmids that carry foreign DNA.
Define gene cloning
The process by which a gene of interest is located and cloned (copied) to produce multiple copies.
Define recombinant DNA
DNA that is formed by combining DNA from different sources often from different kinds of organisms.
Making recombinant insulin: two methods of isolating the insulin gene?
- Restriction enzymes are used to cut the human genome into fragments and then a probe is used to isolate the gene.
- DNA primers are used to isolate gene recognising sequences on both side of the gene and then PCR is used to amplify the gene.
Making recombinant insulin: process?
- Plasmid DNA cut using specific enzyme that creates sticky ends.
- Foreign DNA (with gene of interest) cut using same restriction enzyme.
- Insulin gene isolated (from bacterial cells), then inserted the into the lacZ segment of the plasmid DNA. DNA ligase added and joins their sticky ends together.
- Now a recombinant plasmid is formed which carries the insulin gene.
Making recombinant insulin: how to identify host cells with recombinant plasmids?
Process is a form gene cloning
- The recombinant plasmid is incorporated into a bacterial host cells.
- Some of the bacteria I will take up a plasmid. Some will take up regular plasmids, while some will take up the recombinant plasmid.
- Bacteria grown on an agar plate containing ampicillin = bacteria that haven’t taken up the plasmid won’t have the gene for resistance to ampicillin, so they will be killed by this antibiotic. So bacteria that do grow must have taken up a plasmid.
- Many plasmids will have a regular plasmid, but some may have the recombinant plasmid, so they are then grown on another agar plate containing ampicillin + substrate for lacZ enzyme.
- Bacteria that possess the recombinant plasmid with the insulin gene inserted into the lacZ segment will grow and form white colonies. Cells without it have an intact lacZ segment and will produce the enzyme that converts the substrate to a blue coloured product, resulting in the formation of blue colonies.
- Bacteria from the white colonies are cultured on a small scale on a regular agar plate. When there is a high level of expression of the insulin gene, it is moved to a large-scale mass culturing.
How to transfer plasmids into bacterial cells
- Cells are placed in an electric field that shocks them to create holes in their plasma membranes in order for plasmids to enter.
- Heat shock the bacterial cells in order to increase the fluidity and permeability of the plasma membrane of the bacterial cells which increases the chance of uptake of plasmids.
Testing to identify genetic status: Define: Genetic screening Genetic testing Presymptomatic testing
- Genetic screening = a method of identifying individuals who are at risk of a particular disease or disorder.
- Genetic testing = a method of confirming or ruling out the present of a particular disease or disorder.
- presymptomatic testing = a method use to detect gene mutations associated with disorders that appear after birth.
Ethical considerations (pros + cons) for genetic testing?
Pros:
• People can assure themselves if they are at risk to know if they need regular health monitoring.
• Find genetic factors that could be passed to children, which can help plan pregnancy and use of prenatal testing.
Cons:
• May cause anxiety and stress if tested positive for a late-onset disease, particularly one with no treatment.
• Test results may be inconclusive or difficult to interpret, creating uncertainty for the individual.
Define DNA profiling
DNA profiling is a technique for identifying DNA from different individuals based on short tandem repeats (STRs) or microsatellites.
*Australia now uses 24 STR markers and AMEL (Amelogenin)
Which DAN is used in DNA profiling?
- Nuclear DNA = from the chromosomes, inherited from both parents, undergoes recombination, present as two copies only.
- mitochondria DNA = exclusively inherited from the mother, does not undergo recombination, present in high numbers in somatic cells.
Short tandem repeats (STRs)
- Define
- How many bps?
- In which DNA?
- STRs = chromosomal sites where many copies of a short DNA sequence are repeated over and over
- 2-5 bps long
- Nuclear DNA
Define DNA fingerprinting
DNA fingerprinting is a technique for identifying DNA from different individuals based on variable numbers of tandem repeats of short DNA segments near the ends of chromosomes.
*minisatellites, 9-80 bps are repeated 10s or 100s of times.
Why microsatellites rather than minisatellites?
Micro:
• Far more sensitive
• Requires smaller amount of DNA - can use PCR to amplify
• quicker
• can use more than one locus = more precise
• can use fluorescent labels = can print in colour = easier to read
