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DNA Poly III
3'-5' exonuclease proofreading activity
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dam methylase
Methylates GATC sequences in bacteria DNA, for a template for mismatch repair.
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Mut H, S, and L
- Mut S recognizes mismatch sequence
- H and L excise error
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Post replication repair/homologous recombination repair (T-dimers)
- RecA directs repair off template strand
- DNA poly and ligase fill gap
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SOS repair
Mutagenic DNA replication in response to damage to other repair mechanisms. A last ditch effort.
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Photoreactivation repair
- UV light activates protein the cleaves TT dimers,
- prokaryotes only, photolyase
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Base Excision Repair
- DNA Glycosylases recognize chemically altered bases and cut them out creating AP sites
- AP endonuclease creates a kink from the AP site which is then corrected
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Nucleotide excision repaeir
- Uvr (UvrA UvrB UvrC)
- clip out a total of 13 nucleotides to repair lesion.
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Xeroderma pigmentosum
in humans disruption of Uvr genes, causes excessive freckling and ulcers when exposed to UV light.
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Double strand break repair
- Homologous recombination repair: RAD52 in G2 phase of cells
- Non-Homologous recombination repair: Error prone in G1 phase
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PCR ingredients
- Template
- Primers, (1 for each end)
- DntP's
- DNA polymerase (heat resistant)
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PCR steps
- Denaturation
- primer annealing
- extension
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miRNAs
gene silencing in plants
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Methylation
decrease in gene expression in eukaryotes
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Deamination
causes an increase of AT base pair repeats overtime
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Deamination repair
base excision repair
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Dideoxy chain termination
no hydroxyl group at 3' position causes polymerase termination.
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DNA proofreading
3'-5' by several polmerases
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Mitochondial diseases
In humans the male donates no mitochondria
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Different Mitochondria
Heteroplasmy
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1:1 phonotype petite ratio of yeast
segregational petite
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Segregational mitochondrial diseases
a deletion of a nuclear gene for mitochondrial function
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Suppressive mitochondrial disease
causes wildtype to become mutant,small deletion which enhances proliferation but disable function
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Neutral Mitochondrial Disease
Large deletion, f2 gametes WT and mutant
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Prions
- cause, bovine spongiform encephalitis
- can induce non-mutant proteins to become mutant
- will not affect a host without a gene for the target protein
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Poly-dT oligos
separate rRNA and tRNA from mRNA
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Mouse Homologous recombination
ES cells that contain Neo but not thymadine kinase
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PCR primers
remember, DNA poly transcribes 5'-3' copy off primers(reads 3'-5')
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contig
contiguous sequence: i.e chromosome
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cDNA library
smaller, no introns, made form mRNA
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Microarray expiriment
compares mRNA copy number between two different cells
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Ced
- C. elegans
- Ced3 promotes cell death
- Ced3(-) = no cell death
- Ced9 inhibits cell death
- Ced9(-) = lots of cell death
- Ced9-->Ced3-->cell controlled death
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Laser ddNTPs
5'big sqiggle--->3'little squiggle
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