Part 1
Where does the most common cause of mutation come from?
DNA replication
X-rays, UV light, chemicals, etc.
How do proteins have specific activities?
Because of their shape and chemistry.
What is DNA sequencing used for and what is the most common method?
DNA sequencing used to determine the sequence of an unknown stretch of DNA nucleotide by nulcleotide.
Most common method is dideoxy sequencing(chain termination).
How does DNA polymerase fix a mistake before it becomes a mutation?
Proofreading each nucleotide added 1-by-1.
How does UV damage DNA?
Causes thymine dimers that are covalently bonded to each other, creating a bulge in the double helix; making it difficult for DNA replication or transcription to occur.
Where does point mutations have the potential to directly impact protein function?
In the open reading frame
What is the difference(s) between dideoxynucleotides and deoxynucleotides?
Dideoxynucleotides lack a 3' hydroxyl group, so can't be elongated by DNA polymerase.
Deoxynucleotides has a 3' hydroxyl group, allowing it to be elongated.
What is MMR and what does it do and how.
MMR is mismatch repair that fixes whatever DNA pol missed, does this by taking out a chunk of DNA and matching it correctly.
What are the steps to Nucleotide Excision repair?
1. Enzymes detect irregularity in the DNA structure and cut damaged strand.
2. Enzyme excises nucleotides on the damaged strand.
3. DNA polymerase fills in the gap in 5' to 3' directio.
4. DNA ligase links the new & old nucleotides.
What does PCR stand for and what does it do?
PCR stands for Polymerase Chain Reaction. PCR amplifies and rapidly makes millions of copies of a specific DNA sequence to isolate a certain gene.
What is the ranking of outcomes if there is a mutation in the human genome?
1. No effect
2.Reduction or elimination in gene activity(Loss-of-function mutations).
3. Gain of function
What are the differences in mutations that occur in Germ cells and that occur in somatic cells?
Mutations in Germ cells are heritable and rarer, mutations in somatic cells are not heritable and more common.
What happens in base excision repair?
1. A bad base pair is noticed
2. The bad nucleotide is removed
3. DNA pol and ligase fill the hole with the correct nucleotide.
What are the three steps of PCR?
1. Denaturation(Use heat to unwind the helix)
2. Annealing(cool the solution and use two DNA primers to anneal their complementary sequence on the DNA template strands).
3. Extension(Taq polymerase synthesizes new DNA strands by extending primers).
Describe Pre-implantation Screening
Sperm and egg from parents create embryos in dish.
Can screen embryos for a specific trait(usually any diseased alleles). Then choose which embryos to implant.
No new DNA is introduced, can prevent birth of a child with a disease but cannot cure it.
Describe CRISPR gene therapy
Guide RNA brings Cas9 to a very specific spot
(*ideally) where it cuts the chromosome.
Added DNA can serve as donor for homology-
directed DNA repair; changes seq
can fix a gene (or break it!)
can introduce something entirely different
Can change somatic and germline cells
What are the events of gene expression regulation that happen post-translational?
Name all mutation types we covered
Point mutation
Insertion or deletion of one or two nucleotides
frameshift
silent
nonsense
missense
What is in PCR mix and what is not?
In: Primers, taq polymerase, dNTPs(nucleotides), buffers, template DNA,didNTPS
Out: Helicase, ligase, primase
Describe AAV gene therapy.
Modified virus can bring DNA into human cells (a
“vector”). This DNA could be natural or engineered.
could be functional version of defective gene
could be extra version of something already functional
could be something entirely novel
AAV delivers DNA to nucleus where it can be
transcribed and translated (but not replicated!)
Effect of added DNA is transient (months to years)- no
change to genome of patient, somatic cells only
Describe RTV gene therapy
Modified retrovirus can bring DNA into human cells
and integrate it into chromosome. (a “vector”). This
modified genome will be passed on to daughter
cells.
If cell is somatic, affects that patient only
If cell is germline, passed on to offspring
This DNA could be natural or engineered.
Where it inserts into chromosome is not controlled-
could be disastrously mutagenic. Can be used on
somatic cells or germline