Lab 2 Flashcards
Does our genome change throughout our life?
DNA is subject to constant change
- depurination
- cytosine deamination and guanidine oxidation every 5 days
- 600 events of methylation of adenine per day/cell
How do most DNA repair systems work
Most DNA repair systems use the other strand as a template - advantage of 2 strands
When do most mutations in a cell cycle occur?
During DNA replication
- Template strand isn’t available
What are available repair systems for DNA mutations?
- Proofreading exonuclease during DNA synthesis
- Mismatch repair removes mismatche section
- Polymerases
What are two implications of mutations on human health?
- # of mutations that fathers inherit to their offspring increases with age due to continuous production of germ cells
- Rate of mutation in most viruses is higher than in cells, increase successful variants
What are two ways to do random mutagenesis in the lab?
- Mutagenic bacterial strains using bacterial mutation (higher mismatch)
- Mutagens - introduce plasmids into regular bacteria using toxins that modify DNA
Amplification by PCR
Generate mutations by PCR
1- Cycles of denaturation to separate DNA strands
2- Annealing and priming of small fragments of DNA that are specific to the region we want to synthesize
3- Extension - polymerase extends primers, forming new DNA strand and desired part of DNA
What are the 4 ways we can modulate the mutation rate of PCR
1- Using an error prone DNA polymerase
2- Increasing Mg/replacing Mg for Mn
3- Altering the ratio of dNTPs so that there is an imbalance between them
4- Adding mutagenic nucleotide analogues (mutagenic PCRs)
What is best enzyme/polymerase when wanting to incorporate mutations?
Taq polymerase
- proofreading exonuclease have highest fidelity
Explain increasing Mg and introducing Mn
- Mg helps to modulate changes
-Mn increases rate of incorrect incorporation by increasing dNTP incorporation
