Genetic engineering Flashcards
Natural function of restriction enzymes
breaks DNA molecules into shorter lengths at specific nucleotide sequences
Explain the formation of recombinant DNA molecules
Target gene and plasmid vector/DNA ends are ligated together using DNA ligase to form recombinant plasmid/vector/DNA
Properties of plasmids that allow them to be used as DNA cloning vectors
Bacterial plasmids are small, circular, double-stranded DNA molecules
1. low molecular weight – plasmids are more resistant to damage during plasmid extraction or manipulation
2. presence of at least one selectable marker
3. Presence of at least one selectable marker
4. Presence of origin of replication - to make more copies of the DNA
Steps for DNA cloning
- Gene isolation
- Gene splicing
- Bacterial transformation
- Selection and confirmation
Gene isolation
option 1: obtain a copy of the gene by isolation genomic DNA (for organisms whose genome has not been sequenced like mammoth)
option 2: obtain a copy of the gene by isolating mRNA (for organisms whose genome has been sequenced like human) – reverse transcription is performed on mRNA isolated from organism using reverse transcriptase, followed by polymerase chain reaction to obtain the isolated ___ gene insert
–only tissues that expresses the gene will contain the relevant mRNAs
–Advantage: gene sequence isolated does not contain “nonsense” DNA sequences
Gene splicing
The DNA of plasmid is cut open using restriction enzyme - the restriction enzyme cuts the DNA at specific sequence of bases aka restriction site, leaving specific exposed DNA sequences, aka sticky ends (restriction digestion)
Sticky ends with the same sequence as those on the plasmid are also created using the same restriction enzyme - Sticky ends will allow for complementary base pairing between plasmid and DNA of interest(DNA ligation)
DNA ligase catalyses the formation of a phosphodiester bond between 2 DNA strands
The foreign DNA and plasmid are ligated to form a recombinant plasmid . (not all plasmids will take up a foreign DNA, some plasmids will self-ligate to form non-recombinant plasmids)
Bacterial transformation
Recombinant DNA/ vectors/ plasmids have to be returned to be bacterial cells but cell wall is a barrier to entry
Bacteria are described as transformed once the plasmids are taken up
RP/ligation mix is transformed into appropriate bacterial (host) cell made competent by calcium chloride treatment with heat shock/ electroporation – tiny pores are temporarily created to allow for the entry of recombinant plasmids
Selection
selection: identification of transformed bacteria colonies that harbour recombinant plasmids
Following the transfer of competent E. coli (or wtv host) cells containing recombinant DNA, the transformation mixture is plated onto a selection medium containing the appropriate antibiotic.
The marker gene is expressed only when the desired gene has been successfully inserted into the genome of the host.
Confirmation (RD)
Restriction digestion method - very time consuming and only a small number of clones can be screened at one time
- plasmid DNA is first extracted and digested with the appropriate restriction enzyme(s)
- agarose gel electrophoresis is used to visualise the resulting DNA fragments
- Non-recombinant plasmids will yield a single DNA band corresponding to the size of the vector DNA
- recombinant DNA will yield two bands - one corresponding to the vector DNA and the other corresponding to the insert DNA
Confirmation ( PCR)
Polymerase Chain Reaction - quicker
- Plasmid DNA is first extracted and PCR is performed using primers that anneal to either side of the Multiple Cloning Site (MCS) on the plasmid vector
- PCR product size will vary according to the size of the insert DNA located within the MCS
-Agarose gel electrophoresis is used to visualise the resulting DNA fragments
- Non-recombinant plasmids will yield a small product corresponding in size to the MCS region between the primer binding sites
- Recombinant plasmids will yield a larger product consisting of the insert and residual MCS sequences.
Final Confirmation
Final confirmation using DNA sequencing – expensive and time-consuming
- performed using an insert or a vector-specific primer
- sequence data can then be used to interrogate sequence databases to confirm the identity of the cloned DNA insert and make sure that the recombinant plasmid contains the gene-of-interest.
What are Genetically-modified organisms? (GMO)
Genetically-modified organisms: an organism that has its DNA modified by artificial means (the only DNA involved in the organism’s DNA – by turning off a gene or turning on one
What are transgenic organisms?
An organism that has foreign gene(s) from an organism of a different species introduced into its DNA
- A type of genetically-modified organism as it has been modified at the genomic level BUT using DNA from a different organism
All transgenic organisms are GMOs but not all GMOs are transgenic
Hunger and malnutrition
- WHO has stated that hunger and malnutrition are the greatest threat to the world’s public health.
- GMOs have a part to play in worldwide improvement of agricultural production by increasing the quantity and nutritional value, and by reducing loss due to pests and weed growth
Example:
Golden rice– rice modified with daffodil genes to have more beta-carotene, which the body converts to Vitamin A
Social implications of gene technology
advantages for the society:
- improved, cheaper medicines
- improved food supplies, improved nutritional quality of foods
- a cleaner environment
- improved treatment of genetic diseases
Disadvantages for the society:
- unexpected reduction in crops due to ecological disturbance
- farmer made dependent on specific varieties, needing a fresh seed annually and expensive fertilisers
- reduced natural biodiversity resulting in a reduced possibility of new varieties arising
- a reduced effectiveness of antibiotics as more bacteria become resistant
