Gene Technology Flashcards
What is gene transfer?
The insertion of a gene from one source into a different organism
What are the four main steps of gene transfer?
1) Obtain the donor DNA
2) Insert the donor gene into vector
3) Transforming the recipient cells
4) Selection of transformed microorganisms
What enzymes cuts DNA into fragments?
Restriction endonucleases
How do restriction endonucleases work?
-cut DNA double strand
-by hydrolysis reaction
-at a specific base sequence called a recognition sequence
What are the 3 ways to obtain the required donor gene?
-> cut the gene out from DNA
-> make DNA from mRNA
-> direct synthesis of a gene using a gene machine
Explain how to cut the gene out from DNA (gene transfer)
-restriction endonucleases cut chromosomal DNA into fragments by cutting the DNA at recognition sequences
-restriction enzymes will produce fragments with blunt or sticky ends
-sticky ends allow for joining of different DNA sections by complementary base pairing
-gel electrophoresis separates out the DNA fragments and a DNA probe will identify the fragment required
Explain how to make (double stranded) DNA from mRNA
-mRNA is isolated and extracted from cells actively expressing the desired gene
-reverse transcriptase catalyses the production of a single strand DNA copy (cDNA) from mRNA
-DNA polymerase is used to make a double stranded DNA strand
(-sticky ends may be added to the gene)
What is a vector?
A delivery tool to carry the gene into the host cell where it can be replicated and expressed
What are the possible vectors the donor gene can be inserted into
-bacterial plasmids
-bacteriophages
How is the donor gene inserted into a vector?
Bacterial plasmids
-the plasmid is cut open using the same restriction endonuclease as used to cut out the donor gene (so they will have complementary sticky ends)
-the gene and plasmid are mixed and joined together using DNA ligase to form a recombinant molecule
-DNA with foreign DNA inserted into it is called recombinant DNA so the plasmid is a recombinant plasmid
Bacteriophages
-a bacteriophages with a gene spliced into its DNA will inject its DNA into a bacterium, transferring the recombinant DNA
How is the bacterial cell transformed?
-the plasmids and bacterial cells are incubated with calcium ions and subjected to heat shock treatment
-once the bacteria have taken up the gene, they are transformed
What are the 3 possible bacterial outcomes after transforming the cells
The bacteria
-fail to take up any plasmid
-take up non-recombinant plasmid
-take up recombinant plasmid
What can be used to identify recombinant or transformed bacteria?
Marker genes
e.g antibiotic resistance and fluorescent marker genes
Explain how you would select the transformed microorganisms
-grow bacteria on an agar plate containing ampicillin (only bacteria with a plasmid are ampicillin resistant and grow)
-replica plating is used to transfer the ampicillin resistant bacteria to an agar plate containing antibiotic tetracycline/ not containing tetracycline
-all bacteria grows except those with resistance to tetracycline (THE RECOMBINANT PLASMID)
-plates are compared to identify the bacteria that are missing from the plate with tetracycline
-the missing colonies are the transformed bacteria
What are transformed bacteria examples of
-> genetically modified organisms (GMOs)
-> genetically engineered microorganisms (GEMs)
Give examples of products produced by genetically engineered microorganisms
-insulin
-human growth hormone
-enzymes
-adhesives
-lung surfactant protein
-interferon
Explain the benefits of using GEMs to produce insulin
-can be mass produced
-the insulin is identical to hormone naturally produced by humans
-more effective and less time consuming then extracting from dead animals
-non-human insulin can cause allergic reactions
How are genes inserted into plant cells?
-‘Gene guns’ -> microscopic pellets covered with DNA with the donor gene are shot through the cellulose cell wall into plant cells (can be used with species resistant to the bacterium)
-Agrobacterium tumefaciens
-> desired gene is inserted into a Ti plasmid
-> recombinant plasmid introduced to Agrobacterium tumefaciens
-> host plant cell must have cellulose cell wall removed by cellulase
-> bacteria enter plant at a point where it has been damaged and then integrate bacterial DNA (from a plasmid) into the DNA of the plant
Why are genes introduced into plants?
-higher crop yields, increased variety, better food quality
-produce pest and disease resistant crops (reducing pesticide use)
-cultivate GM Crops that grow in unfavourable conditions
How is a gene inserted into animal cells?
-liposomes-> DNA enclosed in a lipid vesicle which cross the phospholipid bilayer of the membrane easily
-electroporation-> disrupting the cell membrane (making more permeable) by the use of high voltage treatment
-viruses-> insert donor DNA into cells (eg adenoviruses or retroviruses)
-inject DNA-> insert donor gene directly into the fertilised eggs of the animal (all cells in the animal will contain recombinant DNA)
Uses of Genetically Modified viruses
-killing human cancer cells
-treating bacterial infections
Why are genes introduced into animal cells?
-encourage faster growth rate and better food quality traits
-produce substances of medical and pharmaceutical value
-use as models in human disease research
What is gene therapy?
The insertion of a functional allele into cells affected by a particular genetic condition
What may cause a genetic disease?
Absent or faulty genes
What are the 2 approaches to gene therapy?
- Somatic-cell gene therapy
- Germ-line gene therapy
Explain somatic-cell therapy
-targets only affected tissues
-that are easily accessible (eg lungs)
-can be used at any stage of an individuals life
Explain germ-line gene therapy
-involves replacing replacing the defective gene(s) in the fertilised egg
-all the cells of the developing individual are normal
-no issue targeting affected tissues in body
-defective gene will not pass to future offspring
What approach to gene therapy is not permitted
Germ-line
