Lecture 9 - Mutations and Molecular Medicine, PCR

Question 1

Type of cancer therapy (2)

Answer 1

• 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)

Question 2

Replication in-vitro: Polymerase Chain Reaction (PCR)

There are _ components:

Answer 2

five

1. DNA sample
2. Primers: single-stranded DNA sequences with flanking region at 3’-end
3. dATP, dTTP, dGTP, and dCTP (deoxynucleotides)
4. DNA polymerase
5. Buffer with salts/ions

Question 3

Taq polymerase (2)

Answer 3

• 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.

Question 4

PCR cycle (3 steps)

Answer 4

1. Denaturation (95oC)
   2.Annealing (60oC)
2. Elongation (72oC)
   Repeat from step 1

Question 5

Gel Electrophoresis (2)

Answer 5

• 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

Question 6

Mutation (defn)

There are __ types of mutations

Answer 6

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

Question 7

Mutations allow for ________ and ______ _________.

Answer 7

evolution; genetic diversity

Question 8

Point mutation is a change in a ______ ________. There are ___ types of point mutations.

Answer 8

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)

Question 9

Silent mutations are mutations that _____ affect protein function because the mutation occurs in a ___-_______ region or the mutation codes for the ______ _______ _____.

Answer 9

don’t; non-coding; same amino acid

Question 10

Loss of function mutations produce a _____ that is not _______. They are usually _______. An example is the _____ ____ ______.

Answer 10

protein; functional
recessive
sickle cell anemia

Question 11

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 _____.

Answer 11

new function
dominant
p53; cancer

Question 12

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.

Answer 12

Phenylketonuria (PKU)
enzyme; phenylalanine hydroxylase (PAH)
conversion of dietary phenylalanine; tyrosine
enzyme; phenylalanine; phenylpyruvic acid

Question 13

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 ___ _______.

Answer 13

protein; cannot
heat-sensitive
colder; functional
not functional

Question 14

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.

Answer 14

amino acid
similar amino acid.
Sickle cell anemia; loss of function

Question 15

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.

Answer 15

stop codon
shortens; coding
may not.

Question 16

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.

Answer 16

insert; delete
3; codons
reading frame
non-functional

Question 17

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 ______ _________ _____ (___).

Answer 17

extensive changes
Deletions
Duplications
Inversion
Translocation
Down syndrome; 21
Translocation; chronic myelogenous leukemia (CML).

Question 18

Chronic Myelogenous Leukemia (2)

Answer 18

• cancer caused by proliferation of white blood cells

- chromosomal translocation places a kinase abl under regulatory elements of bcr (constitutive active)

Question 19

Gleevec - medicine genetics

Answer 19

• 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.

Question 20

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 ______.

Answer 20

16,000
80%
enzymes; DNA bases
enzymes; cancer

Question 21

There are two ways mutations occur (2)

Answer 21

1. Spontaneous mutations: occur with no outside influence. Mistakes are performed by DNA polymerase and imperfect meiosis.
2. Induced mutations: occur due to outside agents or mutagens like UV radiation.

Question 22

Two examples of spontaneous mutations:

Answer 22

1. Base tautomerization: tautomers are constitutional isomers (same molecular formula). Tautomerization causes the bases to not be able to pair with the appropriate base.
2. DNA metylation: increases the likelihood of mutations. When methylated cytosine loses an amino group it becomes thymine.

Question 23

Two examples of induced mutations:

Answer 23

1. 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.
2. Radiation: ionizing radiation causes the creation of free radicals that changes bases into forms not recognized by DNA polymerase.

Question 24

Errors in DNA bases can be corrected in three ways:

Answer 24

1. 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.
2. 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.
3. Excision repair: cells have some enzymes that scan the DNA for damaged bases.

Question 25

Mutations in DNA repair proteins: ________ ______ (___ gene) is part of the machinery that recognizes DNA damage in ______ _____ ____. Mutations in this protein can lead to the _____ disease of _______ _______. It is characterized by _____ ______ and ______ of _____ at an early age.

Answer 25

Xeroderma pigmentosum (XPC gene); nucleotide excision repair.
recessive; xeroderma pigmentosum.
sunlight sensitivity; development of carcinoma

Question 26

A cell can become cancerous due to _________ of _____ _____. An example is ____ ____ ________.

Answer 26

accumulation of genetic mistakes

colon cancer biogenesis.

Question 27

Tools used in medicine genetics: Restriction enzymes
Restriction enzymes are evolved in _____ to fight against ___ _______ by _________. They break the ___ ______ ____. Each enzyme _____ and ___ a _____ _____ called _______ ____. An example of a restriction enzyme is _____.
Restriction enzymes are used to _______ _________. They ____ ____. Specific restriction enzyme can distinguish between _____ and ___-_____ versions of the gene. Use _____ ________ to separate after digestion.
Three results from use of restriction enzymes:

Answer 27

bacteria; DNA insertion; bacteriophages (viruses)
DNA backbone bonds
recognizes; cuts; specific sequence; restriction site
EcoRi

identify mutations
isolate DNA
mutant; wild-type
gel electrophoresis

1. number of fragments
2. sizes of the fragments
3. relative abundance of fragments, indicated by intensity of the band.

Question 28

DNA fingerprinting (2)

Answer 28

• combines the idea of using restriction enzymes with highly polymorphic regions of the DNA to identify people. Highly polymorphic regions of the DNA will make it unlikely for two people to have the same pattern after digestion.
• The FBI uses 13 STR loci in its combined DNA Index System (CODIS) database.

Question 29

Identification of new species (2)

Answer 29

• using restriction enzymes, new species can be identified (DNA barcodes)
• compare pattern of digestion of cytochrome oxidase gene for identification of new species.

Question 30

Genetic screening

Answer 30

test to determine if a person has a genetic diseases

- prenatal, newborns, asymptomatic people

Question 31

DNA testing (2)

Answer 31

• PCR is used to amplify a small sample of DNA

- hybridization can be used to identify if amplified DNA has a particular mutation/genotype.

Question 32

Genetic Medicine

Answer 32

modify genetic disease phenotype

• gleevec is used to selectively inhibit the enzyme that is overexpressed in CML (chronic myelogenous leukemia); example of enzymatic inactivity.
• PKU can be prevented with low phenylalanine diet; dietary changes
• type-1 diabetes is cured by administration of insulin; supply of missing protein

Question 33

Gene therapy

Answer 33

supply the missing gene by insertion of a new gene.

use adeno-viruses to insert the desired gene

Question 34

Photoreceptors - blindness

Answer 34

• addition of a third opsin in adult red-green color deficient primates was sufficient to produce trichromatic color vision behavior.