Genetic Engineering Flashcards
What are some of the reasons for genetic engineering?
-making crop plants resistance to disease, pests and herbicides
-making livestock resistant to diseases and pests
-improving the yields from crop plants and livestock
-modifying animals to make human proteins for medicines that are difficult to obtain by other methods
Explain the first step of genetic engineering and the 3 different ways this can be achieved
1) Obtaining the required gene
a. Using mRNA
-obtain the mRNA for the gene, then add some free DNA nucleotides and the enzyme reverse transcriptase. This enzyme catalyses the opposite reaction to transcription, allowing the conversion of the mRNA into a single strand of DNA. The DNA then needs to be made double stranded, done by adding DNA nucleotides, primers and DNA polymerase
b. Using a DNA probe
-make the DNA sample single stranded (heat) then add the probe which is complementary to the gene you are looking for. Then once the gene is found it can be cut out of the DNA using restriction enzymes
C. Using an automated machine
-if you know the nucleotide base sequence of the gene you want, the gene can be synthesised artificially in the lab using an automated machine, adding DNA nucleotides and DNA polymerase
What can you do to get multiple copies of the gene once you’ve obtained it?
Use the PCR technique- makes millions of copies in a short space of time
Explain step 2 in genetic engineering. Include what a vector is with examples, the role of restriction enzymes and the role of DNA ligase
2) The formation of recombinant DNA; inserting the chosen gene into a vector
-a vector is something that can carry a gene.
-examples are plasmids, viruses, liposomes (synthetic vesicle) and Ti plasmids (tumour inducing, used for plant cells)
-when inserting a gene into a vector, restriction enzymes are used to cut open the vector DNA and to cut the required gene out of any DNA it is attached to (they are little molecular scissors)
-the recognition sites for restriction enzymes in the DNA are 4-6 bp long, usually palindromic, some leave blunt ends, some leave sticky ends
-The staggered ends are more useful as the exposed bases that stick out are used to join pieces of DNA together
-when 2 sections of DNA have been cut with the same restriction enzymes, giving them complementary sticky ends, they can be rejoined using the enzyme DNA ligase which seals the sugar phosphate backbones together
-when the pieces join this is called Recombinant DNA
What are marker genes and what are they used for?
-when the chosen gene is inserted into the plasmid, at least one ‘marker gene’ is also inserted at the same time
-they allow the desired gene to be ‘tracked’ so that you can check if it’s been taken up by the host cell. This is important as you need to know which cells have successfully been genetically modified before growing them on a large scale
Explain how and why 2 marker genes are often used instead of just one, giving examples
-the first marker gene is use to identify which bacteria have successfully taken up a plasmid. It is an antibiotic resistant gene, so transformed bacteria are identified by growing on a medium containing the antibiotic.
-the second marker gene distinguishes between the bacteria that have taken up an empty or recombinant plasmid. When a recombinant plasmid is formed, the desired gene is inserted in the middle of the second marker gene, making it NON-FUNCTIONAL. so bacterial cells that express the second marker gene do not contain the recombinant plasmid
-the second marker gene has easily identifiable phenotypes e.g produce a fluorescent protein or resistance to a different antibiotic
Explain step 3 of genetic engineering, including the 3 different methods used to carry this out
3) Getting the vector into the recipient cell
-the vectors containing the chosen gene are added to a solution containing the chosen host cell.
-DNA doesn’t easily cross cell membranes, but various methods can be used to make it easier for DNA to cross the cell surface membrane on the recipient cell
a. Heat shock treatment = subject bacterial cells to alternating hot (42 degrees) and cold (0 degrees) in the presence of calcium chloride ions. This makes the walls and the membranes of the bacterial cell more porous, allowing the uptake of the recombinant vector
b. Electroporation = a high voltage pulse is applied to the host cell to disrupt the cell surface membrane
c. Electrofusion = electrical fields are used to introduce DNA into host cells
Explain step 4 of genetic engineering
4) identifying which host cells have been successful in taking up the new gene
-this is where the marker genes help to make sure you know which cells have the desired gene before growing them on a commercial scale
Give reasons why prokaryotes are often genetically engineered to make human products rather than using eukaryotes
-simple cell structure = easy to manipulate their DNA, removing and inserting plasmids
-fast growing = reproduce asexually by cloning every 30 minutes in optimum conditions
-cheap to grow = can be grown in large scale fermenters with very cheap nutrients
-no ethical issues = higher organisms / animals would cause ethical problems
How are plants commonly genetically engineered?
-using a soil bacterium which causes tumours in healthy plants, containing plasmids called Ti plasmids
-the desired gene is placed inside a Ti plasmid along with a marker gene
-plasmid then inserted back into the bacterium, which is added to a dish containing plant cells, allowing the plasmid to pass directly into the plant cell
-the infected plant tissue then forms a callus (tumour- mass of GM plant cells)
-each callus cell can be grown into a whole new plant in the right conditions
Explain how genetically modified plants can also be formed through electrofusion
-2 different plant cells are fused together by applying tiny electric currents to their cell membranes causing their cell and nuclear membranes to fuse and from a hybrid cell, containing useful genes from both parents
-the hybrid cell will be a polyploid because 2 diploid cells have been fused. It can be tested with plant hormones to grow into a whole new plant
Give some strengths and weaknesses of using genetic engineering compared to selective breeding
STRENGTHS:
-can alter single specific characteristics rather than crossing 2 whole organisms
-much quicker results as seen in 1 generation
-can transfer genes between different species which would never normally be possible (only members of same species can breed together)
WEAKNESSES:
-unknown side effects of transferring genes between different species
-often seen as unethical by public
-expensive procedures with low success rates
How are animals usually genetically engineered?
Using liposomes and viruses
