Toward what electrode would the DNA would move to in gel electrophoresis?
positive charge because DNA is negative
List 4 components that must be added to do a restriction digest
2. restriction enzyme
What is the UV sensitive dye used to stain DNA and why is it dangerous?
1. Ethidium Bromide
2. It's a mutagen that can cause DNA mutations
What restriction enzyme was used to cut DNA for identification of sickle cell gene? and it's recognition site?
If the mutation is present for sickle cell, what size would the DNA fragment be?
1.401 kb or 1401 bp
What gene encodes the protein that functions as a chlorine channel?
CFTR - cystic fibrosis transmembrane conductance regulator
What protein chains make up human hemoglobin? what is the name of the protein that is most commonly mutated?
1. Beta chains and Alpha chains
2. HbS and HbC
What is the trinucleotide repeat associated with HD? and how many repeats are considered necessary for disease status?
2. repeats > 42
What is the relationship between repeat number and age of onset and severity?
1. ↑ repeats = earlier age onset and ↑ penetrance (severity)
What restriction enzyme is used to identify HD? and what is the DNA recognition sequence?
1. Sst I
2. GAGCT C - sticky ends
What is the size of target sequence if its a normal HD allele? and what is target sequence if its a mutated allele?
1. Normal - 830 bp or .830kb
2. Mutant - 1967 bp or 1.967kb
How do you calculate Chi² value?
1. Find Expected value of total observed volume.
2. Plug in Expected values and observed values for each group of crosses.
3. Add all crosses together to determine Chi²
What is the null hypothesis?
H₀ = there is no real significance between the expected and the observed.
If you reject H₀, then there is a significance between E and O.
If you fail to reject H₀, then there is no real significance between E and O.
When do you reject your null hypothesis?
You reject null hypothesis if you fall outside of the .05 level of significance which deviates away from H₀ and values within .05 do not deviate from H₀.
What is the best fit straight line in a standard curve?
The best fit straight line is the slop of the migration pattern in DNA size and distance from gel electrophoresis.
How do we create a standard curve for DNA fragment size in an agrose gel? What are the minimum components of standard curve graph?
1. Our marker gel is used for the basis of our standard curve. Measure the distances of bands and record the distance relative to band size.
2. Plot the points on the standard curve graph and draw the best fit straight line in our data set.
Why do we use a standard curve?
A standard curve is used to estimate the migration distances between known and unknown molecules relative to the data in which the curve was based on.
What two things determine why and how DNA fragments run in an agrose gel? how does size affect the migration of DNA fragments?
1. the concentration of agrose in the gel and size of DNA fragment.
2. Smaller fragments will run further and faster than larger DNA fragments.
How do you determine the correct amount of agrose to add to buffer to get a specified % of agrose gel?
1. 1% agrose gel = 60g, buffer = 60mL
2. % (60g) = amt of agrose to get specified % of agrose gel.
ie) .8% (60g) = .48g
Why do we run a standard ladder along with our samples?
To compare the expected (standard) with our observed.
How will intact DNA versus highly fragmented DNA run on your gel? what will bands look like?
1. If DNA is intact, then there would only be 1 band of a few bands depending on the organism. ie) humans = 23 bands, bacteria = 1 band.
2. If DNA was highly fragmented, then there would be lots of bands present that look like a smear.
If you were to take intact DNA from a eukaryotic organism and break into fragments...how would that run on a gel and what would the bands look like?
Eukaryotic organisms contain millions and billions of bp in their genome. There would be many broken fragments and the gel would look like a smear.
If lambda DNA was cut with a restriction enzyme, how would the gel appear?
1. lambda DNA is a virus with 49bp in a linear shape. When cut, there would be few bands visible in the gel.
Why is alcohol used in the spooling process?
1. DNA is insoluble in alcohol but soluble in H₂O.
2. When DNA is spooled, DNA will become highly fragmented
What are restriction enzymes and what do they do? What are recognition sites?
1. Endonucleases are enzymes found in bacteria that can recognize specific DNA sequences and cut break them apart. Typically used as a defense mechanism against phage.
2. recognition sites are specific DNA sequences that restriction enzymes find and break apart.
How do yo determine the number of cut sites in a DNA fragment based on the number of bands that you see in your agrose gel after restriction digestion? Is this determination different if the DNA is circular?
1. Number of bands - 1, n-1
2. since Lambda DNA is linear, there would be n-1 cuts.
3. If DNA were circular there would be n cuts. ie) 7 bands = 7 cuts.
List the 3 steps in a standard PCR cycle. What is happening in each step?
1. Denaturation: DNA is heated to 90-95⁰C for H bonds to break
2. Annealing step: DNA is cooled to 45-65⁰C (depending on Primer) for primer to anneal to DNA
3. Extension: heat DNA 70-72⁰C for TAQ polymerase to replicate DNA.
What is a PCR primer, why is it necessary? How many primers do you need to do a standard PCR?
1. Primer identifies specific DNA sequences (STRS and VNTRS) and tags them by attaching to either end of target sequence. They also act as primers for DNA replication.
2. For one standard PCR, you would use 1 primer.
What are the advantages of PCR over cloning to amplify DNA?
1. quick and easy
2. direct visualization without southern
3. old, degraded, and small segments of DNA from anything can be used.
What type of DNA are we looking at when we do DNA fingerprinting?
we are looking at VNTRS and STRS which contain nucleotide repeats that are polymorphic in humans.
Li Fraumeni Syndrome
1. dominant inheritance pattern of specific cancers
2. onset of cancer is early in age
3. contains multiple primary tumors.
What is the "two hit model" of gene inactivation in familial cancer?
In a germ line mutation, 1 somatic mutation will inactivate both alleles.
In normal genes,2 sequential mutations are required for inactivation of suppressor gene.
Characterize the gene, it's chromosome location, protein size, and its function?
1. short arm on Chromosome 17
2. 53000 polypeptides
3. Cell regulator- works by regulating transcription.
What are the 3 domains of the protein?
1. amino terminus region that activates transcription
2. central region with "hot spots"
3. carboxyl region
Why is p53 important in cancer?
1. p53 regulates cell growth. It halts the production of mutated cells. Inactivation of gene will yield in dysfunctional production of p53 protein.
2. p53 is a non-specific tumor suppressor and can cause the development of any type of cancer.
What are the characteristics of LFS?
1. cancer is diverse and has lots of variations
2. any type can develop
3. any tissue can be involved
4. onset is early