Genetic engineering in medicine Flashcards
Describe the role of Restriction Fragment Length Polymorphism (RFLP) in sickle cell disease.
Sickle cell anemia arises from a single nucleotide muataion (A>T). This mutation is in the MsII enzyme restriction site, and MsII will not cleave there any more after the mutation.
This is seen in a southern blot of DNA cut w/ MsII and hybridized with a ß-globin probe. The sickle cell DNA fragments will be larger.
Elaborate on detection of mutations by allele-specific oligonucleotide probes.
You isolate DNA, amplify it by PCR, and expose samples of DNA (put on nitrocellulose paper) to a probe that binds to a specific allele and another probe that binds to a different allele.
An example is cystic fibrosus when you use 2 probes to test to see if family members have CF or are carriers, or don’t have it at all.
How is Variable number of tandem repeats VNTR used in forensics?
VNTR are hypervariable regions that contain sequences that are repeated in tandem a variable number of times. Cleavage of an individual’s genomic DNA w/ a restriction enzyme produces 2 fragments containing this region. The length of the fragments depends on the # of repeats they contain. This is variable for person to person which can help in identifying suspects in crime scenes.
How would you produce Human Growth Hormone?
insert cDNA for hGH into a bacterial expression plasmid (eg E.coli) and hGH will accumulate inside the bacterial cell. You can isolate hGH from the periplasmic space by rupturing the bacterial cell.
How would you produce human insulin?
start w/ full length of cDNA for human insulin, clone out A and B segments, and produce A and B protein fragments separately in bacterial cells. Combine them and they will refold and then oxidize the cysteines to create active insulin.
How would you produce Human Tissue Plasminogen Activator (tPA)?
You would isolate the cDNA and insert it into a plasmid containing the dihydrofolate reductase gene (dHFR) and transfect it into MAMMALIAN CELLS (tPA is too large and complex to be done in bacteria).
Cells are grown in methotrexate, which destroys plasmid-negative cells, and tPA is secreted into the cell culture medium and purified.
How would you produce Hepatitis B vaccine?
HBV surface protein is glycosylated, and bacteria lack glycosylation enzymes so you would produce this protein in yeast, then isolate the cells by centrifugation. and isolate the protein afterwards.
What is the diff b/w in vivo vs ex vivo gene therapy?
In vivo: therapeutic genetic material is introduced directly into the patient
Ex vivo: the target cells are first removed form pt, and exposed to gene therapy vector in the tissue culture dish, then the genetically modified cells are returned back to the patient
Describe how to use a retrovirus for gene therapy.
You replace the viral genes with the RNA copy of the therapeutic gene. The retrovirus will enter the cell, and create DNA by reverse transcripase, and the DNA will incorporate itself into the host-cell genome, and the host will naturally produce the therapeutic protein.
