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13 Cards in this Set

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Germ Line Therapy
Transforming the genes in the egg or sperm
Somatic Gene Therapy
Transform the bad cells and introduce the wild type gene into a specific tissue within the whole organism
Example: A kid with cystic fibrosis
Lets transform the cells in the lungs by inserting the wild type copy of CFTR but inserting the gene into a specific tissue to change somatic cells
Electroporation
Take naked DNA and using an electrical pulse the DNA will enter the cell
Microinjection
A single cell is held by a vacuum and a needle punctures the membrane to insert DNA
Biolistics
Dehydrate DNA and then use a gun to shoot DNA into cells
Viruses
Transduction: Viral transfer
Transfection: Transform animal cells
Viruses are infectious, it is very easy for the virus to inject DNA
They are good vehicles to transform animals cells into foreign DNA
Function and Structure of AAV
Attaches to the cell and the AAV genome integrates on a unique spot on chromosome 19
The virus does not replicate by itself; it requires co-infection which provides the proteins/molecules for it to reproduce
Usually replicates with adeno-virus or herpes
Structure
Single stranded DNA with 7 genes
Has inverted terminal repeats at both ends which play a role in replicating the genome and insertion at the specific locus at chromosome 19
Restriction Endonuclease
Provides the CFTR gene fragment
Cuts up the DNA
Mechanism of the PCR
We want to break the hydrogen bonds in DNA by raising the temperature
The PCR will raise the temperature to 94 degrees for a minute or two and the strands will denature
We now have specific single stranded DNA primers
Lower the temperature to 45 – 65 degrees for 15 seconds so the primer can anneal to the strand with hydrogen bonding
We then have DNA polymerase to extend the DNA in the 3’ direction at a temperature of 72 degrees
pBluescript
Resistant to ampicillin, so if you mix it with a bacteria the bacteria will show resistance to ampicillin ... A bacteria with this plasmid would grow and without it would die
Origin of replication to replicate itself when its in the bacteria
Lac Z to create B-galactosidease to metabolize sugars
Lac Z and the multi cloning site of pBluescript
Lac Z spans the gene but is interupted by a multi-cloning site, which is a place where lots of enzymes can be used to clone things into that site
If you clone something in, you interrupt the lac Z gene, rendering it non functional
What is X gal and when does it turn blue?
X gal is a sugar
It turns blue in the presence of b-galactosidease
If the lac Z gene is interrupted and b galactosidease isnt made, X gal turns white
Bacteria on the same plate can turn blue or white. So what?
White bacteria are harboring plasmids that have the cloned gene
Blue bacteria means that lacZ isn't interrupted so there is no piece of DNA in the cloning site of pBluescript