RESTRICTION ENZYME AP LAB

Pre-Lab → Due Block, 2/8 or 2/9

  • Read pp. 111-123 in AP Lab Manual
  • Read over the story of the murder of Dr. Eugene U. Gene and protocol attached.
  • Put a title in your lab notebook and answer questions 1 & 2 below. Complete sentences are not required.
  1. The table below shows the restriction sites for some widely-used restriction enzymes. Construct the table in your Lab Notebook and use the description of the restriction site provided in the table to determine the information requested for each DNA sequence/restriction enzyme combination listed below. (Please Note: You do not need to copy the nucleotide sequences listed below the table.)

Restriction Enzyme / Restriction Site / Number of Base Pairs in Restriction Site / Number of Sites in DNA Sequence / Resulting Number of Fragments / Size of Resulting Fragments / End Type(Sticky, Blunt)
Eco RI / G / AATTC
CTTAA / G
Bam HI / G / GATCC
CCTAG / G
Hae II / GG / CC
CC / GG
Hind III / AA / GCTT
TTCG / AA

  1. A certain restriction enzyme digest results in DNA fragments of the following sizes: 4400 base pairs, 2200 base pairs, 1600 base pairs, and 600 base pairs. Sketch the resulting separation electrophoresis to scale. Indicate the starting point, positive, and negative electrodes. Explain your sketch.

THE UNFORTUNATE MURDER OF DR. EUGENE U. GENE

Part A - Suspect List Genetic Analysis

The following is information about evidence found at the crime scene and related information about each of the suspects.

  • Every student at the table must complete each suspect analysis using probability calculations (no Punnett squares!) and principles of Mendelian genetics.
  • Please provide a key for each trait in II and III.
  • For I, II, and III,
  • Record the suspect name:
  • Record the parental cross for each suspect
  • Calculate the probability in fraction form of the described traits occurring in the suspects using probability rules of multiplication and addition.
  • If more than one genotype is possible for the parent, use the genotype that would give the greatest likelihood of the trait occurring in the suspect.
  • You must show your work and the probability for every trait, even if the probability for a particular trait is 0.
  • Please circle or highlight each answer
  • When you have completed your probability calculations for I, II, and III, calculate the total probabilityof having all the traits described in the perpetrator for eachsuspect.

……………………………………….

  1. A drop of blood was found at the scene of the crime. Suspects have tested the blood and they have identified it as O negative.

A background check on Lucille Allele determines that her father has O+ blood and her mother has type A+ blood.

Documents show that Gloria Gamete’s parents both had O blood. Gloria’s mother is Rh negative. Her father is Rh positive but it states on his birth certificate that his mother was Rh negative.

There is no record of Poindexter Punnett’s parents’ blood type, but he has a brother with O- blood and a sister with AB+ blood.

Dr. Ormendel confirms that his parents both had type B+ blood.

Records verify that both of Dee Anay’s parents had O+ blood, however she has a brother who is Rh negative.

  1. A hair was found at the scene of the crime that detectives have linked to the murderer. Chemical analysis show the true hair color is blonde, a recessive trait. None of the suspects has blonde hair, but detectives believe the murderer may be chemically altering his/her hair. In addition to hair color, detectives are also analyzing the texture of the hair. Hair texture is a trait that exhibits incomplete dominance. The hair found at the scene is wavy. Detectives have done some background work with family photos and interviews.

Family photographs show Lucille’s father with wavy, brown hair and her mother with beautiful, blonde curls.

Gloria’s father was bald at a young age but school pictures show a young boy with straight brown hair. Gloria’s mother was well-known for her long brown waves.

Photographs of Poindexter’s deceased parents show a family history of curly hair … both of his parents had curly brown hair. In addition, his brother was nicknamed “Curly” because of his brown, curly hair and his sister has curly, blonde hair.

Dr. Ormendel’s parents both have straight, brown hair; however Dr. Ormendel’s sister is a natural blonde.

Dee Anay’s parents both have wavy, blonde hair.

  1. When detectives interviewed Dr. Gene’s staff, several people mentioned they hear the sound of a door opening and closing, followed by three sneezes. Sun-sneezing is an inherited trait that produces an uncontrollable urge to sneeze in sunlight or bright light. All suspects deny sun-sneezing tendencies, so detectives once again do some background work.

*Refer to the pedigree below to determine the path of inheritance for the trait of “sun-sneezing”.*

There is no family history of the sneeze reflex in Lucille’s family and neither of her parents are sun-sneezers.

Gloria’s parents are both sneezers.

Although no one has any clear recollection of Poindexter’s parents sneezing, his brother is a known sun-sneezer but his sister is not.

Dr. Ormendel’s mother often teased his father for his sun-sneezing habit because she did not have the habit.

Dee’s parents are both sun-sneezers, although her sister is not a sneezer.

Which suspect(s) can be ruled out???

THE UNFORTUNATE MURDER OF DR. EUGENE U. GENE

Part B – DNA Analysis

Setting Up the DNA Electrophoresis Gel:

  1. Inoculate each well of the gel using the following procedure.
  • Place an unused disposable pipette tip on the micropipette.
  • Prepare to load the micropipette by pushing down the plunger completely. Insert the micropipette through the foil into the first DNA sample. Release the plunger slowly and withdraw the sample into the pipette.
  • Carefully lower the micropipette through the buffer into the first well of the electrophoresis gel. Load the gel by pushing down on the plunger carefully, releasing the contents of the pipette into the small well completely without touching the gel itself.
  • Withdraw the pipette. Discard the tip.
  1. Repeat for each DNA sample.

Running DNA Electrophoresis:

  1. Load the gels into the specified chamber. Make sure the wells are closest to the negative terminal of the chamber.
  2. After all gels are loaded in the electrophoresis chamber, slide the cover on. Make sure the electrode terminals are making good contact.
  3. Set the power source to 70-75 Volts.
  4. Plug in the power source, turn it on, and look for tiny bubbles forming on the sides of the chamber.
  5. Allow the DNA to migrate for approximately 35-45 minutes or until the tracking dye has moved 3.5 to 4 cm from the wells.
  6. After electrophoresis is completed, turn off the power, unplug the power source, disconnect the leads, & remove the cover.

Staining the Electrophoresis Gel:

  1. Pour the buffer from the plastic staining chamber into the designated container. Rinse the chamber with water so it can be used as a staining chamber for the gel following the electrophoresis procedure.
  2. Use masking tape to label the staining chamber with your period number-table number.
  3. Add ~75 mL of distilled water to the staining chamber.
  4. Use the spatula or gloved hands to carefully remove the gel from the electrophoresis chamber.
  5. Gently slide the gel only into staining chamber. Rinse plastic tray with water and return to the cart.
  6. Float a piece of Insta-Stain, blue side down, in the staining chamber. Allow gel to stain overnight.
  7. After overnight incubation, remove the gel from the chamber. Rinse carefully but thoroughly with dH2O. Blot gently.
  8. Use the slide box to identify the DNA banding pattern for each lane. Examine the DNA found at the crime scene and compare the banding patterns of the suspects’ DNA.
  9. Empty the staining chamber into the sink. Remove the masking tape label. Rinse and dry the chamber.

Data/Data Analysis

The chart below is a key showing the identification of each of the samples used for the electrophoresis procedure.

Specimen / Identification
A / DNA from Crime Scene cut with Enzyme 1
B / DNA from Crime Scene cut with Enzyme 2
C / Gloria Gamete cut with Enzyme 1
D / Gloria Gamete cut with Enzyme 2
E / Dee Anay cut with Enzyme 1
F / Dee Anay cut with Enzyme 2
X / Practice Loading Dye
  1. Use a ruler to draw the gel in your lab book.

Sketch the gel pattern of the DNA bands. Include a key.

  • If your gel was not usable, sketch another table’s gel.
  • Document the gel you used and explain why you were unable to use your gel
  1. According to the results, which suspect can be ruled out?
  2. Does the DNA analysis correlate with the genetic analysis you did in Part A? Explain.

Lab Analysis Questions – Answer the following questions in complete sentences in your Lab Notebook.

  1. What is the charge on a DNA molecule? Explain the reason for the charge and its importance in this procedure.
  2. What is used to fragment the DNA in an electrophoresis procedure? From where are they obtained? Why are they produced in nature?
  3. The initial samples are suspended in a blue dye solution … Describe two purposes of the dye.
  4. What is the basis for separation of the DNA fragments; in other words, what determines how far a fragment moves?
  5. The segment of DNA shown below has restriction sites I and II, which create restriction fragments A, B, and C. Which of theelectrophoresis gels shown below best represents the separation and identity of these fragments? Justify your answer.
  1. Assume a genetic disorder is homozygous recessive. A DNA gel electrophoresis is performed on an individual to determine if he/she is a carrier for that disorder.
  2. As compared to a normal result, how would the results of a carrier differ?
  3. How would the results of an individual with the disorder compare with a normal electrophoresis pattern?
  4. Examine the DNA electrophoresis results shown below. Is the individual tested the father of the child? Explain your answer.
  1. A represents a linear chromosome, B is a circular bacterial chromosome. Please Note: HindIII and HaeIII are two different names for the same restriction enzyme
  1. If you were to digest both strands (A and B) with EcoRI, how many DNA fragments would result for each? Explain.
  2. What enzyme(s) would you use to cut DNA A, keeping the “Red” gene intact?
  3. In order to insert the “Red” gene into the bacterial chromosome and use the “Blue” gene as a loss of function reporter gene, which restriction enzyme would you use? Explain.
  4. Could HaeIII be used? Explain.