Genetics Lab Final Review

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Genetics Lab Final Review
2013-06-25 21:30:12
Genetics Lab

Lab review for final in genetics BIO-207
Show Answers:

  1. How do you get your expected values?
    Ratio multiplied by total observed value
  2. What is the Chi square formula?
  3. 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)
  4. How do you determine degrees of freedom?

    *n is the number of phenotypes
  5. What is the null hypothesis?
    What we are testing. It is real or significant difference between the expected and observed results
  6. When do you reject your null hypothesis?
    When your calculated Chi square number is high or higher than the table Chi square number.
  7. 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
    • it

    Take that answer and divide by the expected number

    Add all the answers from here to get your Chi square number
  8. 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
  9. 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.
  10. 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
  11. Is DNA soluble in water?
  12. Is DNA soluble in isopropanol?
  13. Is DNA soluble in ethanol?
    No and ethanol works better than isopropanol in experiment
  14. 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
  15. Is DNA sticky?
    Yes because it stuck to glass rod during extraction
  16. Spooling
    Method that partially purifies, concentrates and isolates high molecular DNA from a aqueous solution
  17. 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
  18. 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

    Analyze bands
  19. What does PCR stand for?
    Polymerase Chain Reaction which is a process that produces large amounts of copies of a specific piece of DNA
  20. List the 3 steps in a standard PCR cycle. What is happening in each step?
    • Denaturation:   C unwind duplex by breaking hydrogen bonds
    • Annealing:  C allows primers to anneal
    • (A-T ->/ G-C -> )

    Extension:  C (optimal temp. for polymerase) polymerase makes DNA
  21. What’s unique about Taq polymerase?
    Its heat stable
  22. 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
  23. What are the advantages of PCR over cloning to amplify DNA?
    Quick, easy, can use degraded DNA, only need small sample
  24. 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
  25. What may happen if the annealing step temperature is too low?
    The annealing will be non-specific which will produce numerous bands
  26. 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
  27. Protocol: PCR
    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
  28. What type of gene is p53?
    A tumor suppressor gene
  29. Why is p53 important in cancer?
    They found that 50% of all cancers have a mutated p53 gene
  30. What familial cancer is associated with a germ line (reproductive cell – sperm/ova) mutation in p53?
    Li-Fraumeni syndrome
  31. What are the characteristics of Li-Fraumeni
    syndrome (LFS)
    • 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
  32. What was the test that was done on the samples that you ran on your gel in lab?
    p53 test
  33. 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
  34. 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
  35. 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
  36. 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
  37. Individual can inherit 3 different combinations
    of genes:
    Two normal genes

    Normal gene and a mutated gene (carrier)

    Two mutated genes (affected) will pass a mutated gene to next generation
  38. Protocol:How to run electrophoresis:
    • Dissolve agarose powder into boiling buffer
    • solution

    Cool to 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
  39. How many bands should you expect?
    1= homozygous

    2= heterozygous
  40. What sizes will bands be?
    You refer to the ladder
  41. The genetic mutation that leads to Sickle Cell (base substitution)?
    A to T
  42. Sickle Cell:Name the restriction enzyme used
  43. Sickle Cell: What DNA sequence does MstII recognize?
    • 5' C|CTGAGG 3'
    • 3' GGACTC|C 5'
  44. Sickle Cell:The size of our DNA fragment in kilobases (kb) before we cut it?
    1.702 kb
  45. Sickle Cell:If the mutation is present what size will our DNA fragment be in kb?
    1.4 kb
  46. Sickle Cell:If the mutation is not present what size will our DNA fragment be in kb?
    1.2 kb
  47. Cystic Fibrosis: What cellular ion transport is abnormal (name the ion)?
    (Cl-) chloride ion
  48. Cystic Fibrosis: What body secretion is abnormal?
  49. Cystic Fibrosis: The most common mutation is called?
  50. Cystic Fibrosis: The most common mutation is the deletion of what specific DNA sequence?
  51. Cystic Fibrosis:What amino acid is deleted in the disease protein?
  52. Cystic Fibrosis:What enzyme was used and what does it recognize?
    BamHI = 5' G|GATC|C
  53. Huntington's Disease: What is its mode of inheritance?
    Autosomal Dominant
  54. Huntington's Disease:Whats the trinucleotide repeat?
  55. Huntington's Disease:How many repeats are considered necessary for disease status?
    42 to 100+ copies
  56. 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
  57. Name 2 other trinucleotide repeat disorders?
    Fragile-X syndrome and Myotonic Dystrohy
  58. Huntington's Disease:What restriction enzyme is used?
  59. 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)
  60. Huntington's Disease: Whats the size of our target sequence (bp) if its a normal HD allele?
    ~1000 bp
  61. Huntington's Disease: Whats the size of our target sequence (bp) if its a mutated HD allele?
    ~2000 bp
  62. Huntington's Disease: What indicates a person has the disease
    the individual shows ~2000 bp fragment either in the homozygous or heterozygous state