Biotech 2
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| What is genetic modification also called? | Genetic engineering.
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| To assemble recombinany molecules what enzymes are used to cut the DNA? | Restriction endonuclease enzymes.
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| What enzyme is rejoins the DNA fragments? | DNA ligase.
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| Cells containing foreign recombinant DNA are termed? | Transgenic or genetically modified.
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| What do restriction endonucleases enzymes do? | Cut a phosphodiester bond with a specific DNA sequence.
These enzymes are a natural defense mechanism that bacteria use.
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| Restriction endonucleases recognise specific sequences termed? | Recognition sequences (usually between 4 and 8 nucleotides long)
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| Recognition sequences are often palindromes, what are these? | Sequences which read the same in either direction e.g. 5' to 3'.
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| What are blunt ends? | When there are no nucleotide overhangs.
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| How do sticky ends transiently rejoin? | By base pairing (hydrogen bonding) but the original cut must be repaired to complete the sugar-phosphate backbone to make a stable recombinant DNA molecule.
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| What enzyme repairs the orginal cut to complete the sugar-phosphate backbone? | DNA ligase.
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| What does DNA ligase do? | It is involved in DNA replication where it jons Okazaki fragments. It ligates breaks in dsDNA molecules by reforming the phosphodiester bond.
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| What ligase enzyme is commonly used in molecular cloning? | Bacteriophage T4 ligase.
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| What joins together sticky ends after restriction digestion? | DNA ligase. It can join together any pieces of DNA provided that they have complementary sticky ends. This means that it can be used to circularise a piece of DNA after it had been linearised by restriction digestion.
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| How are complementary sticky ends created on both pieces of DNA? | By cutting them both with the same restriction enzyme.
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| Can DNA ligase also ligate blunt ends? | Yes, but at a much lower efficiency than it ligates sticky ends.
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| What is thermostable DNA polymerase used for? | To amplify the DNA fragment that contains the gene of interest using PCR.
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| Why are prokaryotes especially suitable for genetic modification? | As they are capable of hosting plasmids.
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| What are plasmids? | Plasmids are circular dsDNA molecules that are separate from the chromosomal DNA and are capable of autonomous replication using the bacterial cell machinery.
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| What must a plasmid contain in order to replicate? | An Origin of replication (Ori) sequence.
This is a sequence where DNA polymerase binds and opens up the DNA in order to start replication.
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| What must a plasmid contain to be useful as a cloning vector? | It must contain a number of convenient restriction enzyme recognition sites so that the gene of interest can be inserted and ligated.
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| What name is given to the restriction sites in man-made cloning plasmid vectors that are usually clustered together? | Multiple cloning site (MCS).
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| Apart from an ori sequence and the multiple cloning site vectors must contain a? | Selectable marker and/or a reporter gene.
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| What usually is a selectable marker? | An antibitoic resistance gene that makes it possible for cells that have taken up the recombinant DNA to survive when grown in the presence of antibiotic.
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| What does a reporter gene do? | Causes cells that have taken up the recombinant DNA to either change colour or emit light (in visible or UV spectrum).
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| Antibiotic resistance genes used in plasmids as selectable markers include? | Ampicillin (amp), carbenicillin (Cab), Chloramphenicol (Cam), Kanamycin (Kan), Rifampicin (Rif) and tetracycline (Tet).
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| *What contains resistance against tetracycline and ampicillin?* | pBR322
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| What are some common reporter genes? | Firefly luciferase gene - cells emit light
Jellyfish Green fluorescent protein GFP - cells fluoresce under UV light
enzyme β-galactosidase - can cause bacterial cells to turn blue if grown on media containing substrate X-gal.
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| What makes it possible to distinguish cells that have taken up the recombinant DNA from the ones that have not? | Selectable makers and/or reporter genes.
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| How big is plasmid pBR322? | 4361bp in size. It contains both Amp^r and Tet^r genes.
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| In the absence of any insert, cells transformed with pBR322 will be resistant to what? | Amp and Tet.
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| If an insert were cloned into the SalI site then what would be lost? | Tetracycline resistance would be lost from the plasmid.
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| Transformed cells plated on medium containing tetracycline would be unable to what? | Grow. Only plasmids without any insert could grow.
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| Plasmid pUC19 is *2686 bp* in size, what does it contain? | Amp^r
LacZ
A multiple cloning site within the LacZ gene with restriction sites for 10 restriction endonucleases.
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| β-galactosidase is the enzyme that catalyses the breakdown of what in the lac operon? | Lactose.
In molecular biology it can be used as a reporter gene.
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| When using pUC19 what is used as the substrate? | X-gal, an analogue of lactose.
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| Which colour do colonies containing pUC19 with an intact lacZ gene appear when grown on media containing X-gal? | Blue.
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| Insertion of foreign DNA into any of the cloning sites in the LacZ gene causes insertional inactivation of lacZ. X-gal cannot be broken down and the colonies appear which colour? | White.
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| What does blue-white screening allow? | Selection of colonies carrying plasmids with insert DNA.
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| What does the addition of ampicillin to the medium do? (pUC19) | Prevents growth of clones without plasmids.
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| If the recombinant DNA is to be transcribed and translated into protein by the host prokaryotic cell the cloning vector -must- posses what? | A suitable promoter sequence for the gene of interest in addition to the ori, MCS, selectable marker/reporter gene.
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| What is a promoter sequence? | A DNA sequence upstream a gene where RNA polymerase binds to initiate transcription.
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| What is a strong promoter? | One with a high affinity for RNA polymerase, which therefore maximises transcription.
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| What should an effective vector for gene expression include? | A strong promoter upstream of the cloned gene.
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| Why would a high level of continuous expression of cloned gene be detrimental to the host cell? | It creates an energy drain preventing bacterial growth.
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| Why is an inducible promoter usually a better option than a continuously transcribed strong promoter? | As it can be switched on or off in the cells to allow control of cloned gene expression instead.
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| A good inducible promoter should ensure that? | Recombinant protein expression is kept at a low level until is it induced. Strong enough to produce high levels of expressed protein in the cells after induction. Easy to induce by addition or removal of compounds to the culture media.
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| What are some commonly used promoters? | T7 and lac promoters.
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| Unlike prokaryotes, eukaryotic cells in general cannot contain what? | Plasmids. Yeast is the exception to this rule.
The reason for this is the composition of cell membrane, which in eukaryotes is usually lacking the attachment sites for the plasmid's ori sequence.
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| What is the process called through which plasmids recombinant DNA are allowed to replicate in prokaryotic cells first and after they are purified and used to introduce foreign DNA into host eukaryotic cell? | Transfection.
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| A typical plasmid cloning vector that is to be used in eukaryotes contains? | The ori sequence, selectable marker gene, a multiple cloning site but in addition to these it contains a eukaryotic selectable marker or a reporter gene, eukaryotic cell specific promoter before the gene of interest and a polyadenylation sequence.
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| To insure tissue specific expression of a gene of interest, some eukaryotic cloning vectors contain what? | Tissue specific promoter and a tissue specific enhancer.
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| What is the process of uptake of foreign DNA by a prokaryotic cell known as? | Transformation.
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| Can DNA normally pass through the bacterial cell membrane? | No, as it is a very hydrophilic molecule which cannot normally pass through the membrane.
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| What are bacteria that are capable of allowing the DNA to pass through the membrane called? | Competent cells.
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| Bacteria which are not naturally competent can be made competent for transformation by which methods? | Chemical transformation.
Electroporation.
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| What is electroporation? | It involved exposing the cells in suspension to a short electric shock which appears to briefly open holes or pores in the cell wall/membrane allowing DNA to enter. Different types of electroporator machines are commercially available.
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| What is chemical transformation? | Rapidly dividing cells are suspended in ice cold calcium chloride solution. The DNA to be inserted is then added to a tube, and the mixture is subjected to a brief *heat shock at 43°C*. As well as Ca^2+ any other divalent cation, can also be used.
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| Only cells which have been *transformed with a plasmid* containing an antibitoic resistance hene will be able to do what? | Divide.
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| What does in mean if the bacteria have been successfully transformed? | Those bacteria which contain the plasmid with the antibiotic resistance gene express this gene and grow.
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| What happens to bacteria without the plasmid that do not have/express antibitoic resistance gene as they were not transformed? | The will die.
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| How can yeast cells be made competent? | By using enzymes to digest away their cell wall which allows them to take up recombinant DNA containing plasmids.
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| Yeast cells can also be? | Transfected using *electroporation*.
Transfected using DNA coated microbeads fired at them using a gene gun.
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| Yeast can maintain plasmids with yeast specific what? | Ori.
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| Recombinant DNA can be introduced into a multicellular eukaryotes using... | Electroporation.
Microinjection.
DNA coated microbeads and a gene gun.
Using genetically modified virus as a vector.
Using liposomes.
Using DNA/calcium phosphate co-precipitate.
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| The three different types of genetically modified animals that have been created to date are? | Transgenic animals expressing a foreign gene protein in one or more tissues.
Transgenic animals with a gene deleted/inactivated in on eor more tissues.
Cloned animals.
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| What is germ line modification? | Modifying an animal at embryo stage which creates stable modifications that can be passed on to the animal's offspring.
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| What is somatic modification? | Modifications attempted on a fully grown animal in such a way that only certain tissues are targeted.
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| What is the basis of gene therapy? | Somatic modification.
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| What is much less efficient; somatic modification or germ line modification? | Somatic modification is much less efficient.
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| What is anti-thrombin (AT)? | A small protein that prevents blood clotting by inactivating several enzymes of the coagulation system. It is normally produced by the liver.
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| How can AT be produced by genetic modification? | Through goats milk, which means it is continuously produced and can be extracted without harming the goat.
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| To express AT in goat milk the AT gene in the recombinant DNA construct must have a what? | *Goat mammary cell-specific promoter* before it. Using this type of promoter ensures that the protein will *only be secreted into milk* and not expressed in the other goat tissues.
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| What can happen if the human anti-thrombin protein that is produced contains traces of goat proteins? | It is possible that the *contaminating goat proteins can lead to an immune response* in the patients that receive it as therapy.
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| DNA can be introducted into plants using mechanical methods such as? | The DNA coated micro beads fired from a gene gun.
Microinjection.
Electroporation.
Using *Ti plasmid* based constructs.
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| What is Ti? | Tumour inducing plasmid is a naturally occuring plasmid from a plant pathogen called *Agrobacterium tumefaciens*.
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| Ti plasmid naturally inserts into the genome of which plants? | *Dicotyledonous* plants.
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| Transgenic plants that have to date been generated using genetic engineering techniques are? | Pest resistant plants (e.g. *Bt-soya, Bt-corn, Bt-potato* )
Herbicide resistant plants (*Roundup ready^TM corn*)
Extended shell life fruits (*Flavr savr^TM* tomato)
Nutritional enrichment/synthetic seeds (*Golden rice*)
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