DNA Fingerprinting
Excerpt from SDUHSD Police Report:
Officer Brian Hicks, Badge # A3854F-7
The crime occurred last Friday, in the evening. According to Donna the custodian who happened to be working late that night, as she passed by Mr. Haas’ classroom around 10:00 p.m., she noticed the door slightly ajar but with no lights on. Taking a peek inside, Donna was startled to discover the classroom in complete disarray. Desks were pushed awry around the room, chairs turned over, etc. Mr. Haas’ room was clearly burglarized. Oddly, nothing of value was taken, except Mr. Haas’ prized Usek (Walrus penis). Because the Usek had only minor value on the black market, detectives first suspected other staff members at the school. After questioning the staff, only 3 members were on site, who had some possible motive. Both Principle David Jaffe and Assistant Principle Brian Kern had questioned Mr. Haas just days before about the appropriateness of having a “penis” lying around in class, and had asked him to remove it—to which Mr. Haas refused. Mr. Rik Napora, counselor, was also said to be on campus. According to Mr. Haas, Mr. Napora had recently asked him about possible uses for such a tool at home, as he had recently lost his hammer and had put all of his money towards his car, thus lacking the necessary funds to purchase a new hammer.
According to investigators, the suspect likely left the lights off to prevent being seen by Donna; thus in stealing the Usek, the suspect seems to have run into the corner of the filing cabinet and cut themselves. Although it seems the suspect attempted to clean the blood, some remained. Investigators are pleased to find out from forensic scientists that there was enough blood for DNA fingerprinting and that the results of the DNA tests yet to come will undoubtedly finger the culprit.
FORENSIC ANALYSIS TECHNIQUE RECOMMENDED:
DNA FINGERPRINTING
Electrophoresis has many applications in state-of-the-art molecular biology
Laboratories. One of its most interesting and important uses is in the courtroom using a technique called DNA fingerprinting. The chemical structure of everyone's DNA is the same. The only difference between people (or any animal) is the order of the base pairs. There are so many millions of base pairs in each person's DNA that every person has a different sequence. Using these sequences, every person could be identified solely by the sequence of their base pairs. However, because there are so many millions of base pairs, the task would be very time-consuming. Instead, scientists are able to use a shorter method, by cutting the DNA up into several pieces of different sizes. This is done using one or more restriction enzymes. Just think of restriction enzymes as “molecular scissors” that cut the DNA at specific points in the sequence.
Now a scientist can sort the DNA pieces by size. The process by which the size separation, "size fractionation," is done is called agarose electrophoresis. The DNA is poured into a gel, such as agarose, and an electrical charge is applied to the gel, with the positive charge at the bottom and the negative charge at the top. Because DNA has a slightly negative charge, the pieces of DNA will be attracted towards the bottom of the gel; the smaller pieces, however, will be able to move more quickly and thus further towards the bottom than the larger pieces (see below). The different-sized pieces of DNA will therefore be separated by size, with the smaller pieces towards the bottom and the larger pieces towards the top.
DNA fingerprinting can link a suspect to a particular piece of evidence with an extremely high degree of certainty. For example, a hair found at the scene of a crime and is taken to a forensic laboratory. There the DNA in cells in the root of the hair is extracted and then is cut into pieces using restriction enzymes. An individual’s DNA is as distinctive as a fingerprint since restriction enzymes cut each person’s DNA into different sized pieces. When the cut DNA is separated into its pieces by electrophoresis, the visible end result looks something like the bar code on a super market package. The DNA from the hair root cells can then be compared with the DNA obtained from one or more suspects.
Forensic technique: GEL ELECTROPHORESIS
IN THIS ANALYSIS YOU WILL BE GIVEN A SAMPLE OF DNA LEFT AT THE SCENE OF THE CRIME. YOU WILL SEPARATE THE PIECES, PRODUCING; A PATTERN ON A GEL. FROM THIS YOU WILL BE ABLE TO IDENTIFY THE PERSON GUILTY OF STEALING MR. HAAS’ PRIZED USEK!
OBJECTIVE:
FORENSIC TECHNICIANS will use the method of agarose gel electrophoresis as a tool for separating DNA fragments.
I. BACKGROUND (for intern technicians)
One of the most basic and frequently used tools of the molecular biologist is
electrophoresis. In this experiment, you will be using agarose gel electrophoresis to separate DNA pieces of different sizes. Electrophoresis means “to carry with an electrical currents. What you will observe is that different sized pieces of DNA will be carried different distances by an electric current as the pieces migrate through agarose, a substance derived from seaweed, which you will apply a voltage through. The gel coated slide will be placed in a “gel box” where the gel serves as an “electrical bridge” between two electrodes submerged in a conducting solution. You will place your DNA samples into small wells cast at one end of the gel. Current supplied by electrodes at either end of the chamber creates an electric field across the gel. The DNA and other negatively charged ions will migrate through the pores in the agarose toward the positive end of
gel. The diagram below should prove helpful.
Top _ Charge
+
ELECTROPHORESIS makes smaller DNA fragments move farther/faster through gel than large fragment. It is possible to do gel electrophoresis with DNA because the DNA molecule has an overall negative charge. The negative charge comes from the phosphate groups which alternate with the sugar, deoxyribose, to form the “rails” of the “twisted ladder” of the double helix. The negatively charged DNA is attracted to the positively charged electrode when the current is turned on. The different sized DNA pieces separate because the smaller fragments will migrate farther than the larger ones. After the fragments are separated, you will turn off the electric current, remove the gel and view the DNA pieces under a UV light that reveals the banding pattern of the DNA.
Materials List
Supplies for each lab station: (See page 7 for pictures)
250 ml container with black cap1 micropipettor and tips
Gel electrophoresis box with gel box tray and electrical chords
Plastic “comb”
Power supply
Small empty cup for used tips
Supplies you may get at the teacher station:
4 DNA samples - 3 suspect samples and 1 “crime scene” sampleDNA ladder sample – helps monitor fragment size
Electrophoresis buffer solution (TAE)
Ethidium Bromide (DNA stain – a carcinogen, use gloves!)
Distilled water – to prep the gel and DNA
37°C water bath
Masking tape – to tape the ends of your gel box tray
Sharpies
Paper to trace bands of DNA off gel
Agarose powder- to make your gels (found at side table near scale)
Gloves (1 pair per group)
Procedure for the Electrophoresis - DNA fingerprint analysis
Check the list of materials against supplies at your station.
Check the box off as you proceed through each step!
Preparing the gel – Wednesday
A. Measure 1 gram of Agarose powder and place into 125 ml flask.
B. Add 50 ml of TAE buffer to the flask with powder.
C. Take the flask to the microwave and melt the powder until it completely dissolves. 1st run the microwave for 20 seconds. Then swirl. Then run the microwave again for as many additional increments of 10 seconds as needed until no small particles of powder remain, swirling after every 10 seconds. Do not let the solution boil or your gel will not solidify properly.
Note: The flask gets extremely hot and the solution inside can pop! Use caution, goggles and a heat mit when handling the jar after heating.
D. Allow the flask to cool at your lab station for 10 minutes.
E. While your jar cools, use your micropipette and attach a clean tip. Inject 2 microliters of Ethidium Bromide (EtBe) into the gel solution. (container in tinfoil)
DANGER: Ethidium Bromide is a carcinogen so please use gloves when injecting and when handling the gel in the future.
F. Seal off the ends of your gel box tray by taping the ends with masking tape, preventing any run off.
G. Set gel box tray on a smooth level section of your lab station.
H. Place the LARGE gel comb into the appropriate space so that the “teeth” of the comb are about 0.5 cm from the edge of one side of the gel box tray.
I. Pour the agarose solution into your gel box tray with comb already in place. If after pouring the gel, you see air bubbles, use a micropipette tip to remove them. Bubbles will disturb the DNA. Make sure the solution has cooled before pouring so as to not melt the tape.
J. Allow the agarose solution to cool and solidify for about 15 minutes. During this time, clean out your agarose container with water and return it to the teacher station.
K. After the gel completely hardens, it will appear opaque. Remove the tape.
L. REMOVE COMB by carefully pulling straight up firmly and gently. The indentations left by the comb are the wells into which you will pipette the DNA samples.
M. Clean your comb with water and soap and dry with a paper towel.
N. Take your now hardened gel and place it in a labeled plastic bag. Be careful! The side where the wells were made by the comb breaks off easily and will ruin your experiment! You must wear gloves when handling the gel! It has EtBr in it!
O. While waiting for your gel to dry, practice pipetting 10µl of water onto the letter o in the newspaper. Show your teacher and have them initial here before continuing. ______
P. Once hardened, place your gel in a labeled zip-lock bag.
Preparing the DNA - Thursday
- Each group will prepare 4 different tubes of DNA mixtures—3 suspects and 1 crime scene DNA.
- Each epi tube will have DNA and the restriction enzyme. The amounts and order you insert these items is essential!
- For each color DNA get an empty epi tube (that means a total of 4 per group)
- The items required for each epi tube can be found at the teacher’s station. Do your best to slowly pipette these items into your epi tube, so as to not make air bubbles. Put them into your epi tube in this exact order and at these exact concentrations:
1) DNA 20µL
2) Restriction enzyme 1.0µL
- After combining the DNA and restriction enzyme, gently tap the bottom of the container a couple times to mix. Then spin your epi tube in the centrifuge for 2 seconds. Closing the top will activate the centrifuge.
- Repeat the same procedure for all 4 epi tubes of DNA mixture.
- Make sure your 4 epi tubes are labeled with tape and a sharpie and then insert your tubes into a 37-degree water bath for approximately 30 min. This is the optimal temperature for the restriction enzyme to cut the DNA.
- While your mixture is in the bath, listen to your teacher discuss the background information you need to know for the lab.
I. While waiting practice pipetting 5µL of Phenol Red dye onto the trial gel at the teacher station. Every group member should do this, as each will be loading one lane of the gel.
J. Remove your DNA tubes from the water bath, put in a labeled plastic bag and freeze overnight.
Loading DNA into the gel — Friday
Q. Place the now hardened gel and tray in the gel box apparatus. Again, only handle the gel with gloves and be careful not to break the gel! Place the tray so that the wells are closest to the anode, or negative end (the black end).
R. Pour electrophoresis buffer solution into your gel box (TAE). Pour the solution into one end until it overflows onto the gel and into the other side. Keep pouring until the gel is fully submerged in solution (covered). Check to see that the solution gives a smooth surface over the gel. There should be no “dents” over the wells. Only if dents are present should you add more solution to your gel box.
K. Put a clean tip on your micropipettor.
L. Check to see that the micropipettor is set for 4mL.
M. Pipette 5µL of loading dye into each of your epi tubes. This dye will enable you to see approximately where on the gel your DNA is. Warning: Change tips for each DNA epi tube or you will contaminate the DNA!
N. Once again, tap the bottom gently to mix the solution. Then spin each tube in the centrifuge, 2 seconds.