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					Lab 13 – DNA Detective


Objectives

   Discuss how DNA information could be used or misused
   Analyze a crime scene using Dot Blot


Introduction

         CSI. Crime Scene Investigation. Shows such as this one have highlighted to the general
public that DNA could help you or possibly hold you responsible for certain crimes. You can go
on TV talk shows and have DNA testing to determine if you are the real father of a baby. DNA
spells out our genetic information using nucleotides, and it does this in exactly the same way in
almost every cell of our body. If it were possible to read all 3 billion nucleotide pairs that make
up our DNA, we would find that no other person, with the possible exception of an identical
twin, would have the exact same sequence or order of nucleotides. Like a fingerprint, our DNA
sequence identifies us as distinct individuals.
         Dot Blot analysis is a technique used to determine the presence of DNA, RNA, or
protein. In this lab, we will use Dot Blot to determine if a certain allele (DNA sequence) is
present in a person or at the crime scene. The gene we are testing is DQA1 gene on chromosome
six. The DQA1 gene is one of a large family of genes coding for HLA (human leukocyte
antigen) proteins. These proteins can be found on the surface of certain cells and play a role in
cells distinguishing self from non-self.
         In the human population there are eight different DQA1 alleles. Because two of the
alleles are extremely rare, the dot blot tests for only six alleles: 1.1, 1.2, 1.3, 2, 3, and 4.
Remember, though, that an individual can only have 2 alleles (one from mom and one from dad).
In the dot blot analysis, single-stranded probes (allele sequences) are fixed to a membrane strip.
Then single-stranded sample DNA (victim, crime scene, suspect) is added to the membrane strip.
The strip is then exposed to a stain that will indicate areas where double-stranded DNA is
present. That is, if the victim’s single-stranded DNA sample had the 2 allele, it will bond with
the single-stranded 2 allele on the membrane strip. This area would light up.
         The dot blot membrane strip contains: 1; 2; 3; 4; c; 1.1; 1.2, 1.3 & 4; 1.3; all but 1.3.
Below each number(s) is a probe for that specific allele(s). Below the number 1 are single-
stranded DNA for 1.1, 1.2, and 1.3 alleles. If the sample DNA had 1.1 or 1.2 or 1.3 allele(s),
this probe would light up. Below the number 2 are single-stranded DNA for the 2 allele. If the
sample DNA had the 2 allele, this probe would light up. Below the number 3 are single-stranded
DNA for the 3 allele. Below the number 4 are single-stranded DNA for the 4 allele. Below the
letter c (i.e., control) are single-stranded DNA for ALL the alleles. Below the number 1.2, 1.3,
& 4 are single-stranded DNA for 1.2, 1.3, and 4 alleles. Below the number 1.3 are single-
stranded DNA for the 1.3 allele. Below the area marked “all but 1.3” are single-stranded DNA
for all of the alleles, except 1.3.




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Lab 13 – DNA Detective


13.1 Consider what happened in a small town

        Mavis Grumsby, Grumpy Grumsby to her colleagues at the auditing department of State
Ranch Company, was only slightly disturbed by the letter left under her windshield wiper.
“Meet me at midnight. The usual place.” She crumpled the note and threw it on the ground of
the parking lot. Miss Grumsby thought, “This time, I’m going to put an end to it. He must leave
his wife. There can be only one Mrs. Witherspoon. Enough was enough.”
        Dean Witherspoon, CEO of State Ranch, slammed down the phone in exasperation. Why
wasn’t Mavis answering? He had to talk to her. He wanted to ask her to keep the relationship
hush-hush to save his career.
        Bruno Benson and Muffy Ryerson pulled into the parking space Miss Grumsby had just
vacated. “That was Miss Grumsby! She’s the meanest auditor at State Ranch. She kept
hounding me about the details of my business trip to Chicago last weekend. I’m afraid she’ll
find out that I went to see Bon Jovi instead,” Muffy moaned.
        “How about me,” Bruno muttered. “Grumpy Grumsby is going to tell Witherspoon that
the account that I’m in charge of is $10,000 short. What’s this?” Bruno picked up the crumpled
paper Mavis had thrown down.
        “Sounds like Grumpy Grumsby has a boyfriend. And it’s almost midnight now.” Muffy
yawned. “Maybe a boyfriend will loosen her up.”
        Bruno jammed the paper into his pocket and jumped back into his car. “Shut up, Muffy.
You don’t know anything about this letter, got it? Nothing!” He backed out of the parking spot,
and sped into the night.
        “That’s what you think, lover boy,” Muffy sighed.
        The next morning the entire State Ranch complex was buzzing. “Did you hear?” “Miss
Grumsby was found dead at Miller Park!” “The cops are still there. I heard there was blood
everywhere, and the whole area is roped off.” “I heard they were even cutting up part of the
park.” “Let’s go watch!”
        The cops collected some tissue samples from under the nails of Miss Grumsby.
Apparently, Miss Grumsby fought a good fight until the end. After talking over with
administrators of State Ranch, employees, friends, relatives, and other acquaintances of Miss
Grumsby, the cops narrowed their suspects to three persons: Dean Witherspoon, Bruno Benson,
and Muffy Ryerson.




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     Lab 13 – DNA Detective


     13.1 Motive for killing Miss Grumsby

     Fill in the following table about possible motives that each individual may have had for killing
     Miss Grumsby.

                                                              Motive
     Dean Witherspoon


     Bruno Benson


     Muffy Ryerson




     GENERAL QUESTIONS:

1.      What type of evidence did police gather at the murder scene?



2.      If you were the investigator, what other evidence would you collect?



     13.2 Dot Blot analysis of the DQA1 gene


     Hazardous reminder: Coomasi stain is very toxic. Wear gloves, goggles, and aprons.

1.      You have several samples of DNA: one tube of DNA from the victim, one tube of DNA from
        each of the three suspects, and one tube of DNA from under the nails of the victim.
2.      Obtain a membrane strip, labeled with a specific sample, such as victim.
3.      Dip one end of a capillary tube into the DNA tube labeled with the same sample name as on
        the membrane strip.
4.      Lightly touch each probe region on the membrane strip.
5.      Using tweezers, pick up this membrane strip and dip it into the beaker with the Coomasi
        stain. Be sure that all of the allele regions are exposed to the stain.
6.      Take the membrane strip out of the stain and dip it into the beaker with distilled water.
7.      Take the membrane strip out of the distilled water and place it on a paper towel to dry. You
        can blot gently with paper towels.
8.      Record the alleles found on the membrane strip.
9.      Repeat the above procedure for each of the sample DNA tubes.


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     Lab 13 – DNA Detective


     13.2 Results of Dot Blot

     Fill in the following tables with information obtained from the Dot Blot Analysis. (10 pts)

     In a genotype, there are always two alleles. The alleles may be identical.
                                                                          Genotype
     Miss Grumsby
     Bruno
     Muffy
     Dean


                                                                       Alleles present
     Under Miss Grumsby’s nails


     GENERAL QUESTIONS:

1.      Based on the information above, EXPLAIN who killed Miss Grumsby. (2 pts)




2.      Explain why the probes on the membrane strip are single-stranded DNA and not the double
        helix. (2 pts)




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