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47 Cards in this Set

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