What are the three methods to transfect eukaryotic cell?
3. recombinant viruses (most effective but hardest)
When a area is sensitive to DNase, what does that mean?
It probably is not bound to a nucleosome and potentially an enhancer site bound by transcription factors
How does a reporter gene assay work?
1. link the possible enhancer to a reporter gene that will fluoresce or easily be detected by luciferase
2. Make a mutant gene without enhancer but with reporter gene
3. Transfect into different cells
4. assay reporter activity
What are the 4 PS in medicine?
personal, predictive, preventive, participatory
How does massively parallel DNA sequencing work?
1. DNA sheared to 300-500 bp
2. each length attached to one bead
3. beads dropped in flow cell
4. wells filled with enzymes and dATP, dCTP, dTTP, dGTP sequentially passed through
5. camera takes pics cause of fluoresecent tags
6. computer software sorts out overlaps
how does PCR work?
pcr works to amplify a certain gene for alternative to cloning. primers must be known
1. add excess primers
2. separate strands by heat (95 C)
3. cool to anneal primers (55-65)
4. synthesize new DNA at 72 with heat-stable DNA polymerase
what is hybridization?
Annealing a test DNA to a single-stranded probe to form duplex DNA.
Northern blot detects what?
Western blot detects what?
what is the protocol for southern blot?
1. do gel electrophoresis
2. denature with alkali
3. add probe and Klenow/polynucleotide kinase
4. blot with nitrocellulose paper
What can southern blot be used for?
RFLP, VNTR, DNase hypersensitivity mapping
DNase hypersensitivity mapping can locate what DNA elements?
enhancers and promoters
How would you determine where a DNase hypersensitive site is?
1. add DNase to cells
2. add BamH I restriction enzymes (if you know where it cuts away from gene x)
3. Southern blot w/probe
4. find out if probe hybridizes to small or large fragment. if small, dnase is cutting in addition to BamH I.
RFLP and VNTR: are what?
polymorphisms can be of two types:
1. single base substitutions
2. deletions or insertions altering number of copies of short stretches of DNA.
1. cleaved with special enzyme
2. only certain people with normal code can have the enzyme cleaved, other with mutation will not
3. visualized with southern blot and hybridization with a probe
4. shorter indicate restriction enzyme worked and person is unaffected, those who respond to both pieces are carriers/
RFLP is used in paternity testing , t or f
VNTR, or variable number tandem repeats, tests what?
genealogy, tests location of repeats on chromosomes to see codominant inheritance from mother and father (each variant an allele)
What is used to visualize VNTR?
what are ways to create transgenic mice?
1. select ES cells, electroporate with inserted gene, homologous recombination, select with neomycin, inject into blastocyst, implant in uterus
2. inject gene directly through micro-injection into pronuclei in fertilized egg and implant
What does nuclear transplantation entail?
cells from adult tissue have nuclei, one placed in unfertilized egg and nucleus of egg removed, cell then divides and forms embryo, which can turn into reproductive cloning or therapeutic cloning
iPS stands for what, and what does it entail?
induced pluri-potent stem cells, they are generated by adding transcription factors to turn back the clock, erasing epigenetic marks on DNA and histones and placing them at sites in normal pluri-potent stem cells.
This lays a foundation for research into regenerative medicine.
what is the polymerase used in PCR called?
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