19. Genetic Technology Flashcards
What is genetic technology?
Changing a cell’s DNA to change the proteins synthesised. Nucleotides sequenced and compared with other genomes. Genetic tests can be done to identify carriers of genetic disease, gene therapy can be used for treatment.
Engineering: removing a gene from one organism and transferring it to another - gene expressed in new host.
Define ‘recombinant DNA’.
DNA made from joining pieces from two or more sources, contains lengths of DNA from multiple organisms.
Define ‘transgenic organism’.
Aka. GMO - organism expressing new gene.
Outline how gene transfer occurs.
- Gene identified (cut from chromosome, made from mRNA via reverse transcription, synthesised from nucleotides).
- Copies made using PCR
- Inserted into vector (plasmid, virus, liposome)
- Vector delivers gene to cells
- Cells with new gene identified and cloned.
What are the tools for a gene technologist?
- Enzymes (restriction endonucleases, ligases, reverse transcriptase).
- Genes for identifiable substances (markers).
- Vectors.
What are restriction endonucleases?
Class of enzymes from bacteria which recognise and break down DNA of invading viruses (bacteriophages), by cutting the sugar-phosphate backbone at specific places.
How do restriction enzymes work?
Bind and cut at specific restriction site. Bacterial DNA protected by chemical markers / absence of target sequence.
Backbone cut straight across = blunt end
Staggered = sticky ends (short lengths of unpaired bases, easily form H bonds with complementary sequences on pieces cut with the same enzyme).
How does artificial DNA synthesis work?
No need for template DNA - codons selected, information held in computer and DNA fragments directed. Novel genes (eg. for vaccines) can be synthesised this way.
What are the useful properties of plasmids?
- Found in bacteria (will be taken up)
- Small (easy to manipulate)
- Replicate semi-conservatively (identical copies)
- Replicate independently within bacteria (so gene cloning occurs)
- Can be removed from one bacterial species and be taken up by another (greater flexibility)
- Can be cut at specific locations by restriction endonucleases (for gene insertion).
What are plasmids?
Small, circular pieces of double-stranded DNA, occur naturally in bacteria and often contain ABX resistance genes.
How are genes inserted into plasmids?
Extracted using enzymes which break down cell wall. Naked bacteria centrifuged.
Circular DNA cut using restriction enzyme, this enzyme also used to cut gene - complementary sticky ends.
Open plasmids + DNA fragments mixed, pairing occurs, DNA ligase links sugar-phosphate backbones -> closed plasmid.
Recombinant DNA.
How are plasmids delivered back to bacteria?
Bacteria treated with Ca2+ and cooled, then given a heat shock to increase permeability to plasmids. Some succeed, some take up plasmids without the gene, some don’t take up any.
How are bacteria with rDNA identified?
Spread on agar plates with ABX - eg. gene inserted into sequence coding for resistance, so bacteria with rDNA cannot grow.
How are numbers of bacteria with rDNA increased?
DNA polymerase copies plasmids, bacteria divide by binary fission -> daughter cells with many copies. New gene is transcribed and translated to a product.
How does genetic engineering for insulin work?
mRNA extracted from pancreatic β cells, ones with code for insulin are identified and isolated.
mRNA incubated with reverse transcriptase, used as a template to form single-stranded DNA, DNA polymerase makes complementary strand.
DNA cut with restriction enzyme, sticky ends pair with plasmid, plasmid introduced to bacteria, identified, DNA cloned by binary fission.
Insulin now produced, bacteria grown in fermenters, hormone extracted and purified.
How can mRNA for insulin be changed for different properties?
Insulin analogues.
- can act faster than animal insulin
- can act more slowly to give a background blood concentration of insulin.
How else can rDNA be identified without the risk of ABX resistance?
Enzymes produce fluorescent substances (eg. GFP from jellyfish) - gene inserted into plasmids and UV light used for identification.
β-glucuronidase (GUS) from E. coli - cells incubated with colourless substrates and can make them coloured or fluorescent. Useful in monitoring activity.
What are promoters?
Control regions of DNA to which RNA polymerase binds to start transcription. Appropriate promoters need to be inserted along with desired gene.
Ensures recognition of the template strand, and high gene expression.
Transcription start point = first nucleotide transcribed.
What is gel electrophoresis?
Mixture of molecules placed into agarose gel wells, applying electric field, charged molecules move towards poles. Depends on net charge, molecule size, gel composition, pH.
How does electrophoresis work in proteins?
Buffer solution, charge depends on ionisation of R groups.
Can separate polypeptides produced by different alleles, eg, allozymes, haemoglobin (β-globin has non-polar R group, separated from normal variant).
