Genetics Lab Final Review
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How do you get your expected values?
Ratio multiplied by total observed value
What is the Chi square formula?
How do you calculate your Chi square value?
- 1. Take ratio multiplied by the total observed
- 2. Then subtract that number (expected) from observed
- 3. Then square the answer
4. Then divide that by the expected
- 5. Then = that number (this is the Chi
- square number)
How do you determine degrees of freedom?
*n is the number of phenotypes
What is the null hypothesis?
What we are testing. It is real or significant difference between the expected and observed results
When do you reject your null hypothesis?
When your calculated Chi square number is high or higher than the table Chi square number.
Protocol: Chi Square
- You take ratios 9/16, 3/16, 3/16, 1/16 and multiply these to the total of observed data given = expected number to put into equation
- You take observed number and subtract expected data number from it = take this answer and square
Take that answer and divide by the expected number
Add all the answers from here to get your Chi square number
How will intact DNA verses highly fragmented DNA run on your gel…. What will the bands look like?
If sample barely penetrates gel and moves as a single band then DNA sample primarily contains large pieces of host DNA. If a smearing pattern occurs that moves ahead of main band will correlate to the amount of degradation.
- * Intact (bacteria) DNA = single band
- * Human (eukaryotic) DNA = male: 24 bands female: 23 bands
If we were to take intact DNA from a eukaryotic organism and break it into fragments (with restriction enzymes)….how would that run on your gel…. What would the bands look like?
It will not produce discrete DNA bands they’ll be blurry/smeared. Only way to get clear bands is to run Southern Blot or use small fragments.
Why is alcohol used in this spooling process
(also called DNA extraction)?
To separate the buffer from the DNA and because DNA is insoluble in alcohol
Is DNA soluble in water?
Is DNA soluble in isopropanol?
Is DNA soluble in ethanol?
No and ethanol works better than isopropanol in experiment
This method of alcohol extraction of DNA is used to not only concentrate and extract DNA…what other benefits are there?
It purifies and removes small molecules (salts, sugars, amino acids, and proteins).
It’s convenient way to concentrate nucleic acids
- In extraction of RNA and DNA because in the presence of salt they will precipitate from solutions with high % of alcohol
Is DNA sticky?
Yes because it stuck to glass rod during extraction
Method that partially purifies, concentrates and isolates high molecular DNA from a aqueous solution
Procedure for spooling
Solution of DNA is overlaid with alcohol
- Glass rod is used to mix the two liquids at
- their interface
- DNA precipitates from solution at the mixing
- zone and collects on rod
Protocol: DNA extraction
Freeze 2 ml of DNA solution for 5 min
Add 2 ml ice cold alcohol (let sit for 2-3 min)
Separation will occur, DNA will precipitate
Spool DNA with glass rod (2 min)
Air dry rod for 1-2 min
Add spooled DNA to distilled water in new tube (swirl 1 min)
Microfuge 100 µL (microliters)
Add 10 µL loading dye to sample
Use 0.8% agarose gel for electrophoresis for ½ hr
Stain with ethidium bromide
What does PCR stand for?
Polymerase Chain Reaction which is a process that produces large amounts of copies of a specific piece of DNA
List the 3 steps in a standard PCR cycle. What is happening in each step?
What’s unique about Taq polymerase?
Its heat stable
What is a primer in PCR (why is it necessary)?
How many primers do you need to do a standard PCR?
Single stranded piece of DNA and it has complementary base pairs to the flanking region around the target and 2 primers are needed
What are the advantages of PCR over cloning to amplify DNA?
Quick, easy, can use degraded DNA, only need small sample
What is a thermocycler?
A device that can be programmed to run for a specific amount of time at a specific temperature to amplify DNA sample.
*Amplification is logarithmic
What may happen if the annealing step temperature is too low?
The annealing will be non-specific which will produce numerous bands
What type of DNA are we looking at when we do DNA fingerprinting?
- VNTR's = Variable Number of Tandem Repeats
- STR's = Short Tandem Repeats
Put 20 µL DNA samples into tubes and label properly
Add 20 µL of MMP (Master mix + primers)
Pipet up n down to mix and cap each tube
Add tubes to ice then
- Use thermocycler to perform 3 steps of PCR:
- *35 cycles*
Microfuge for 10 sec
Add 5 µL loading dye to each sample and ladder
Run electrophoresis 35-45 min
Stain with ethidium bromide
View and analyze with UV transilluminator
What type of gene is p53?
A tumor suppressor gene
Why is p53 important in cancer?
They found that 50% of all cancers have a mutated p53 gene
What familial cancer is associated with a germ line (reproductive cell – sperm/ova) mutation in p53?
What are the characteristics of Li-Fraumeni
- Early age onset cancer
- A family that gets a lot of cancers
- Cancer at young age
- Every cell in their body is susceptible to cancer
What was the test that was done on the samples that you ran on your gel in lab?
About p53/mutations/hot spots
- Mutations in the p53 gene are detected in high frequencies in locations called “hot spots”. Majority of the “hot spots” are
- located in the second domain of p53 which is referred to as the central region. There is some correlations in the site of the
- mutation to the tumor tissue
What two things determine why and how DNA fragments run in an agarose gel? How does size affect the migration of DNA fragments (bands on gel)?
The amount of sample used and if it is negatively or positively charged and smaller pieces migrate faster than large fragments
How do you determine the correct amount of agarose to add to buffer to get a specified percentage of agarose gel?
= grams of agarose powder needed
Why do we run a standard (ladder) along with the samples?
To determine approximate size of the dye in base pair equivalent - It’s a reference for band size
Individual can inherit 3 different combinations
Two normal genes
Normal gene and a mutated gene (carrier)
Two mutated genes (affected) will pass a mutated gene to next generation
Protocol:How to run electrophoresis:
- Dissolve agarose powder into boiling buffer
C then pour into mold, let solidify
Submerge gel in buffer in electrode chamber
Add tracking dye to DNA – gives DNA density so it sinks into wells
Heat DNA at
C and load 35 µL into each well
- Cover with lid attach electrodes
- black(neg.) / red (pos.)
Look for bubbles to make sure its running
Allow samples to run 3.5 – 4 cm for proper band separation
Remove from buffer and use UV illuminator to analyze bands
How many bands should you expect?
What sizes will bands be?
You refer to the ladder
The genetic mutation that leads to Sickle Cell (base substitution)?
A to T
Sickle Cell:Name the restriction enzyme used
Sickle Cell: What DNA sequence does MstII recognize?
- 5' C|CTGAGG 3'
- 3' GGACTC|C 5'
Sickle Cell:The size of our DNA fragment in kilobases (kb) before we cut it?
Sickle Cell:If the mutation is present what size will our DNA fragment be in kb?
Sickle Cell:If the mutation is not present what size will our DNA fragment be in kb?
Cystic Fibrosis: What cellular ion transport is abnormal (name the ion)?
(Cl-) chloride ion
Cystic Fibrosis: What body secretion is abnormal?
Cystic Fibrosis: The most common mutation is called?
Cystic Fibrosis: The most common mutation is the deletion of what specific DNA sequence?
Cystic Fibrosis:What amino acid is deleted in the disease protein?
Cystic Fibrosis:What enzyme was used and what does it recognize?
BamHI = 5' G|GATC|C
Huntington's Disease: What is its mode of inheritance?
Huntington's Disease:Whats the trinucleotide repeat?
Huntington's Disease:How many repeats are considered necessary for disease status?
42 to 100+ copies
Huntington's Disease:Whats the relationship between repeat number and age of onset and severity?
The higher the number of repeats the earlier the onset of the disease and the higher the severity
Name 2 other trinucleotide repeat disorders?
Fragile-X syndrome and Myotonic Dystrohy
Huntington's Disease:What restriction enzyme is used?
Huntington's Disease:What is the recognition site for SstI and where does it cut?
- 5' GAGCT|C 3'
- 3' C|TCGAG 5'
- *cut between the C and T on both strands (staggered cut)
Huntington's Disease: Whats the size of our target sequence (bp) if its a normal HD allele?
Huntington's Disease: Whats the size of our target sequence (bp) if its a mutated HD allele?
Huntington's Disease: What indicates a person has the disease
the individual shows ~2000 bp fragment either in the homozygous or heterozygous state
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