Genetic Engineering

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How are restriction endonucleases used?
Endonuclease enzyme will bind to DNA at a recognition site; Enzyme causes a cut at a specific site; Palindrome unpaired bases seen; Produces sticky ends
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How is reverse transcriptase used?
B cells that produce insulin selected; mRNA extracted; Reverse transcriptase used to make DNA from RNA = cDNA as it is made up of nucleotides complimentary to mRNA ; DNA polymerase builds complimentary nucleotides on cDNA to form a new strand
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What is the use of DNA ligase?
Joins two sticky ends of DNA together
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How are plasmids taken up by the bacterium?
Plasmids and Bacterium mixed in a solution containing Ca2+ ions; Temperature is changed and the bacteria become more permeable and taken up the plasmid
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Why are plasmids not taken up by bacteria?
Few bacterial cells take up the plasmid; Some plasmids close up without incorporating the DNA fragment
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How is replica plating used to identify bacteria with the desired plasmid?
Bacteria survive 1st antibiotic; Small sample of original colony transferred to a replica plate in sam emotion as 1st colony; 2nd antibiotic should then kill bacteria suggesting they have taken up the plasmid and lost resistance to 2nd antibiotic
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How are fluorescent markers used to identify bacteria with the desired plasmid?
Gene producing GFP inserted into plasmid; Rapid results from viewing under microscope
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How are enzyme markers used to identify bacteria with the desired plasmid?
Enzyme lactase; Will turn particular colourless substrate blue; Lactase gene replaced with desired gene = bacteria grow in colourless solution
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How is PCR used to for copies of a gene?
DNA strands separated at 95C; Primers added once when temperature is at 45C; Temperature raised to 70C and DNA polymerase copies each strand starting at the primers
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What are the advantages of in vitro gene cloning?
Extremely Rapid; Does not require living cells so no complicated culturing and only amplification
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What are the advantages of in vivo gene cloning?
Useful for introducing gene in another organism as it uses vectors; No risk of contamination; Very accurate; Cuts out specific genes; Produces transformed bacteria that can be used to produce large quantities of gene product
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How are cloned CFTR genes delivered?
Normal CFTR gene inserted into plasmid; Plasmid reintroduced into bacterium for cloning; Cloned copies of plasmids with normal CFTR gene inserted into liposome to allow it to cross cell surface membrane; Liposome introduced to lungs via nasal spray
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outline germ-line gene therapy.
Ensures all cells of offspring develop normally = long term affect and permanent so ethical and moral issues
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Outline somatic-cell gene therapy.
Targets affected tissues = Not present in sperm or egg cells = repeated every few days however targeting stem cells will make it last a lifetime
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Outline the symptoms of cystic fibrosis
Mucus congestion in lungs = high risk of infection due to irremovable disease trapping mucus; Breathing difficulties and less effect O2 exchange; Accumulation of thick dust in pancreatic duct prevent enzymes reaching duodenum= fibrosis cysts;
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How is severe combined immunodeficiency used in gene therapy?
ADA gene isolated isolated using restriction endonucleases; ADA inserted into retrovirus; Grown with hosts cells to make more copies; Retrovirus mixed with patients T-cells; Retrovirus inject normal ADA gene into T-cell; T-cells reintroduced
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Outline the process of genetic fingerprinting
Extraction of DNA; Restriction endonucleases cut DNA into fragments; Fragments separated via gel electrophoresis; DNA fragments transferred from gel to nylon membrane = southern blotting; Radioactive DNA probe added to label fragments; Membrane with
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Card 2

Front

How is reverse transcriptase used?

Back

B cells that produce insulin selected; mRNA extracted; Reverse transcriptase used to make DNA from RNA = cDNA as it is made up of nucleotides complimentary to mRNA ; DNA polymerase builds complimentary nucleotides on cDNA to form a new strand

Card 3

Front

What is the use of DNA ligase?

Back

Preview of the front of card 3

Card 4

Front

How are plasmids taken up by the bacterium?

Back

Preview of the front of card 4

Card 5

Front

Why are plasmids not taken up by bacteria?

Back

Preview of the front of card 5
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