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47 Cards in this Set
- Front
- Back
Function of restriction endonucleases(restriction enzymes)
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Cut-double stranded DNA(at specific sequence) into smaller fragments
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EcoR I
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E. Coli
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Hae III
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Haemophilus aegyptius
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Bgl II
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Bacillus globigii
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bstE II
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Bacillus stearothermophilus
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Define: restriction site
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Specific sequence of bases cleaved by restriction enzyme (normally palendromic - same base sequence when read in 5' -> 3' direction)
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How do u make blunt ends of DNA?
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Restriction enzyme cuts dsDNA in the same spot(ie Alu I)
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How do u make a sticky end?
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Restriction enzyme cuts palendromic sequences w/DNA over hanging (ie EcoRI)
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What is the function of DNA ligase?
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Joins together fragments of DNA(in vivo = repair single stranded breakds, in vitro = joints fragments of ds DNA
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Function of DNA polymerase
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Make copies of DNA
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Function of reverse transcriptase
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Copies RNA -> cDNA
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Southern blot
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DNA
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Northern blot
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RNA
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Western blot
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proteins
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Application of southern blot
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Identify genes, RFLP analysis, carrier detection, DNA fingerprinting)
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Chemical DNA sequencing method
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Maxam-gilbert
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Where is the dideoxy located in the ribose sugar
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3' OH -H thus stops the chain
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Function of PCR
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In vitro method for producing large amiunts of DNA from a target sequence
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Define: transduction
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phage mediated gen transfer into a prokaryotic cell
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Define: transformation
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non-phage mediated gene transfer into a prokaryotic cell
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Define: electroporation
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w/a non-viral vector plasmid
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Define: transfection
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Gene transfer into a eukaryotic cell
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Define: clone of gene
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Means to isolate the DNA that encode s the gene in a pure, readily reproducible form
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Define: genomic library
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A collection of DNA fragments from an organisms ENTIRE GENOME cloned into bacterial colonies
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Define: cDNA library
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A collection of cDNA fragments produced from all the mRNA present in a particular tissue type(corresponding cDNA of a protein). (ie liver cDNA library, pancreatic cDNA library)
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How do u screen for a specific gene in a genomic library?
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Hybridization w/an oligonucleotide probe -> PCR -> immunological screening
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What DNA technique do u use to detect sickle cell disease
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Restriction fragment length polymorphism (RFLP)- detecting a deletion in the Mst II site of the beta-globin gene. Normal gene = 1.1kb and mutant gene = 1.3kb
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What is the DNA method to find cystic fibrosis?
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Use allele-specific oligonucleotide (ASO) hibridization - short synthetic DNA probe that thybridizes to the target sequence
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What is the function of allele-specific PCR amplification (ARMS test)
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PCR equivalent of ASO hybridization(achieves the same purpose in one PCR step) -> use 2 sets of primers: 1st primer for both reactions; 2nd primer in 2 forms - normal and mutant -> only exact complimentary target sequence is amplified
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What is the limitation of PCR?
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>4000 bases
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That is the function of DNA microarray
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Used to detect mutation and to examine gene expression
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Which proteins are made thru recombinant DNA tech?
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1. Insulin, 2. factor VIII, 3. factor IX, 4. human growth hormone, 5. tissue plasminogen activator, 6. interferon, 7. erythropoietin, 8. adenosine deaminase
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How do u make human insulin in E.coli?
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You insert 2 plasmids into two different E.coli cells. 1. A chain DNA plasmid, 2. B chain DNA plasmid -> then when they are excreted the two peptides folds and makes human insulin
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How do we make hep B vaccine?
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Insert HBV plasmid into yeast cells -> to make HBV surface antigen
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Define: gene therapy
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To cure or prevent a disease by modifying an individuals genes or their expression
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Define: gene replacement therapy
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Replacing a mutated gene that causes disease w/a healthy gene
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Define: gene therapy for non-inherited diseases
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Introducing a new gene to help fight a disease
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Define: gene-blocking therapy
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Inactivating a mutated gene that is functioning improperly
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What is the limitation of retrovirus?
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8kb insert max -> integrates into host genome randomly
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What is the limitation of adenovirus
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8kb insert max ->can elicit immune response -> does not integrate into host genome
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What is the limitation of adeno-associated virus
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5kb insert max -> difficult to produce
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What is the limitation of herpes simplex virus?
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20kb inert max -> can cause immune response
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What is the general rule of non-viral vectors?
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Better suited for ex vivo gene therapy vs in vivo gene therapy
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What diseases are found doing ex vivo gene therapy?
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1. SCID, 2. familial hypercholesterolemia, 3. gauchers disease, 4. malignant melanoma
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What is the procedure for ex vivo gene therapy?
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1. Therapeutic gene is inserted into virus, 2. cells from target tissue are removed from patient, 3. cells are grown in large numbers in tissue culture plates -> cultured cells are then mixed w/the virus, 4. the cells are then returned to the patient to replace function lost due to inheritance of mutant genes
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What are the disease that can be cured w/in vivo gene therapy?
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1. Cystic fibrosis, 2. brain, ovarian, neck tumor, 3. hemophilia B, 5. Duchanne muscular dystrophy
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What are the pharm animals?
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Pig = human hemoglobin, goat = human tissue plasminogen activator (TPA)
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