Lecture 9 - Mutations and Molecular Medicine, PCR Flashcards
Type of cancer therapy (2)
- tissue-dependent treatment
- treatment o cancer will depend on the type of tissue affected
- e.g. all types of colon cancer would be treated the same way
- genetic-dependent treatment
- cancer treatment will depend on the genetic mutation
- e.g. chronic myelogenous leukemia (CML)
Replication in-vitro: Polymerase Chain Reaction (PCR)
There are _ components:
five
- DNA sample
- Primers: single-stranded DNA sequences with flanking region at 3’-end
- dATP, dTTP, dGTP, and dCTP (deoxynucleotides)
- DNA polymerase
- Buffer with salts/ions
Taq polymerase (2)
- thermus acquaticus (Taq) DNA polymerase: operates at 72 oC (extension temperature), tolerant of 95 oC (denaturing temperature)
- thermostable polymerases (and the diversity of life on earth) made PCR practical.
PCR cycle (3 steps)
- Denaturation (95oC)
2.Annealing (60oC) - Elongation (72oC)
Repeat from step 1
Gel Electrophoresis (2)
- separates samples by size/charge
- agarose gel for DNA
- polyacrylamide gel for protein
- DNA is negatively charged (phosphate groups)
- DNA moves toward the positive end
- smaller fragments travel faster than big fragments
Mutation (defn)
There are __ types of mutations
Mutations: changes in the nucleotide sequence that can be passed to the next generation
Two
1. Somatic: produced by mitosis and cannot be passed to offspring
2. Germ line: produced by meiosis and can be passed to offspring
Mutations allow for ________ and ______ _________.
evolution; genetic diversity
Point mutation is a change in a ______ ________. There are ___ types of point mutations.
single nucleotide
two
1. Transition: substitutes one purine for another purine; pyrimidine for another pyrimidine (A for G, T for C)
2. Transversion: substitutes one pyrimidine for a purine (A for T, G for C)
Silent mutations are mutations that _____ affect protein function because the mutation occurs in a ___-_______ region or the mutation codes for the ______ _______ _____.
don’t; non-coding; same amino acid
Loss of function mutations produce a _____ that is not _______. They are usually _______. An example is the _____ ____ ______.
protein; functional
recessive
sickle cell anemia
Gain of function mutations produce a protein that have a ____ _______. They are usually ______. An example is the mutation to the ____ tumor suppressor which can lead to _____.
new function
dominant
p53; cancer
Another example of a loss of function mutation is ___________ (___). This disease results from an abnormal _______ called _______ ________ (___). It normally catalyzes ______ __ _____ __________ to ________. Loss of the _____ function causes _______ and __________ _____ to accumulate.
Phenylketonuria (PKU)
enzyme; phenylalanine hydroxylase (PAH)
conversion of dietary phenylalanine; tyrosine
enzyme; phenylalanine; phenylpyruvic acid
Conditional mutations produce a _____ in which the alteration _________ be seen under normal conditions. Example is a Siamese cat whose specific enzyme important for pigmentation is ____-______ and does not work at normal body temperature. At ____ temperature, the protein is _______. At normal body temperature, the protein is ___ _______.
protein; cannot
heat-sensitive
colder; functional
not functional
Missense mutations will substitute one ______ ____ for another. It may not affect protein function if the change is to a ________ ______ ____. A missense mutation that is harmful is ____ ____ ______, which is also an example of _____ __ ________ mutation.
amino acid
similar amino acid.
Sickle cell anemia; loss of function
Nonsense mutations create the formation of a _____ ______. This _______ the protein, if it occurs in the _____ region of the mRNA. If it is near the 3’-end of the mRNA, it may/may not have that much of an effect.
stop codon
shortens; coding
may not.
Frame-shift mutations ____ or ____ bases. Reading frame is composed of every _-base ____, staring with the start codon. This mutation causes an alteration of the ____ ___. It also produces ____-_______ proteins.
insert; delete
3; codons
reading frame
non-functional
Chromosomal mutations are caused by _________ ______ in the DNA. ________ are when a big piece of DNA is missing. ________ are when a piece of DNA is added. ______ are when a piece of DNA is flipped. __________ are when a piece of DNA of one chromosome is attached or exchanged with another. Example is _____ _____ caused by translocation of chromosome ____ in germ line cells. _______ in somatic cells can lead to ______ _________ _____ (___).
extensive changes Deletions Duplications Inversion Translocation Down syndrome; 21 Translocation; chronic myelogenous leukemia (CML).
Chronic Myelogenous Leukemia (2)
- cancer caused by proliferation of white blood cells
- chromosomal translocation places a kinase abl under regulatory elements of bcr (constitutive active)
Gleevec - medicine genetics
- gleevec inhibits an enzyme called Bcr-Abl, a protein kinase that is misregulated in certain cancer cells.
- most normal cell types in the body don’t require this protein kinase and so the drug is non-toxic.
How do mutations occur? In normal circumstances, DNA damage occurs daily - about _______ events per cell per day in humans. About ___% of these are repaired. There are _____ that correct changes in ____ ____. Mutations in these ____ lead to susceptibility to ______.
16,000
80%
enzymes; DNA bases
enzymes; cancer
There are two ways mutations occur (2)
- Spontaneous mutations: occur with no outside influence. Mistakes are performed by DNA polymerase and imperfect meiosis.
- Induced mutations: occur due to outside agents or mutagens like UV radiation.
Two examples of spontaneous mutations:
- Base tautomerization: tautomers are constitutional isomers (same molecular formula). Tautomerization causes the bases to not be able to pair with the appropriate base.
- DNA metylation: increases the likelihood of mutations. When methylated cytosine loses an amino group it becomes thymine.
Two examples of induced mutations:
- Chemicals can alter bases: nitrous acid can deaminate cytosine and covert it to uracil. benzoprene in cigarette adds a chemical group to guanine and prevents base pairing.
- Radiation: ionizing radiation causes the creation of free radicals that changes bases into forms not recognized by DNA polymerase.
Errors in DNA bases can be corrected in three ways:
- DNA proofreading by DNA polymerase. When an incorrect nucleotide is incorporated, DNA polymerase stalls which leaves enough time for the 3’ to 5’ exonuclease to remove a few nucleotides before DNA replication is resumed.
- Mismatch repair: there are proteins that scan for newly synthesized DNA for mistakes. When a mismatch is found, these proteins repair the DNA by cleaving a section of the DNA.
- Excision repair: cells have some enzymes that scan the DNA for damaged bases.
