MCM Final - Tools of Cell and Molecular Biology Flashcards
fluorescence microscopy
use fluorescent tags to visualize a molecule
-absorb light at one wavelength, emit at another
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light microscopy
use light to visualize cells and tissues
in vitro
in glass
in vivo
in the living
medium for cell culture
salts, carbohydrates, amino acids, and vitamins
supplemented by animal serum
-contains growth factors
passaging
process of removing cells from a surface and replating at a lower density
explants
culture a small tissue fragment
-usually in neuroscience research
primary cultures
prepared directly from tissues of an organism
can be homogenous or heterogenous
don’t last very long
immortalized cell lines
over come replicative cell senescence
generated from cancer cells
created by introducing oncogenes
Hayflick Limit
human somatic cells have a limited number of divisions (50-60)
first line of immortalized cells?
HeLa cell line from Henrietta Lacks
hybridoma cell line
used to produce monoclonal antibodies
- infinite quantity of identical antibodies - used in diagnosis and treatment
heterocaryon
cell with two nuclei
process to create hybridoma cell line?
1 create hybrid cell type
-from differentiated normal cell and immortalized cell
2 cells fuse and create a heterokaryon
-use b lymphocytes from mouse and b lymphocyte tumor cells
3 only hybridoma cells survive in the selective medium
4 cells multiply and scan for anti-X antobidy
5 positive clones are a continuous source of anti-X antibody!
antibody structure and function
y shaped
2 Fab domains - antigen binding
1 Fc domain - signaling
one lymphocyte creates one identical antibody in large quantities
polyclonal antibodies
antibodies produced by inoculating an animal against an antigen
-from multiple lymphocytes agains multiple epitopes
Fab Domain
2 on antibody
-allow the binding of antigen
Fc Domain
1 on antibody - allow signaling to occur
primary antibody
bind antigen
secondary antibody
bind Fc portion of primary antibody (bound to antigen)
has a marker than can be identified
immunocytochemistry
stain cells
immunohistochemistry
stain tissues
enzyme-linked immunosorbent assay (ELISA)
marker in enzyme that creates a color precipitate when binds substrate
-used in sensitive tests (infection, pregnancy)
electrophoresis of DNA and mRNA
negative charged phosphate groups
movement based on size of fragment
SDS
covers protein during electrophoresis to give it an overall negative charge
-so it can be electrophoresed
also, need to unfold protein before electrophoresis
Northern Blotting
mRNA detected with labeled oligonucleotide probe
Southern Blotting
DNA sequence detected with a labeled oligonucleotide probe
Western Blotting
aka immunoblotting
-protein detected using primary and secondary antibodies
recombinant DNA
method of producing large quantities of DNA, RNA, or protein
-molecular cloning
source DNA - fragment isolated using RFLPs (restriction endonuclease sites)
cDNA
copy DNA
-need to remove introns if protein synthesized in bacteria
-make cDNA from mRNA using reverse transcriptase
process of creating cDNA
1 make cDNA from mRNA using reverse transcriptase
2 add oligonucleotide linkers with restriction endonuclease cleave sites to the ends of cDNA
3 cDNA ligated to appropriate vector
plasmid vector
DNA molecule capable of independent replication when placed in host cell
- contain: - restriction endonuclease site (same as source DNA) - gene for antibiotic resistance - origin of replication
recombinant vector
combination of source DNA and vector DNA
recombinant proteins
produce large amounts of protein for therapeutic use
expression vectors
in recombinant protein
- require DNA sequences that express protein
- include promoter and start site
gene therapy
introducing normal copy of gene into a cell
retrovirus
carry RNA genome
- reverse transcribe it into DNA - DNA inserted randomly into host DNA - requires host cell to be actively dividing - permanent change
adenovirus
carry DNA genome
- not integrated into host DNA - eventually lost - host cells do not need to be actively dividing
transgenic animals
inject cDNA into fertilized ova
if successful - offspring are chimeric in somatic and stem cells
-next generation will not be
