Whose DNA Was Left Behind?

Whose DNA was Left Behind?

EDVO-Kit #S – 51

**for ~7 groups**

MISD – PreAP Biology

Whose DNA was Left Behind?

DNA Gel Electrophoresis Simulation Lab

PRELAB

Read the background,purpose, and scenario sections of the lab then answer the questions that follow it in complete sentences.

Read the procedure and answer the questions at the end of that section.

In the data section, draw the gel as described so that all you have to do is sketch and color the bands after your have completed the procedure.

BACKGROUND

DNA fingerprinting allows for the identification of the source of a DNA sample, which is very important in many forensic cases. DNA fingerprinting can provide positive identification with great accuracy by matching DNA obtained from a crime scene to individual suspects.

Several steps are involved in DNA fingerprinting. First, a suitable sample must be obtained. Forensic scientists use great care when obtaining evidence from crime scenes so that the DNA will not be damaged. DNA is then isolated from the evidence, such as blood or hair samples. Once the DNA is isolated, it is either digested with special enzymes called restriction endonucleases (restriction enzymes), or submitted to the process of polymerase chain reaction (PCR) to increase the amount of DNA available for testing by exploiting our knowledge of how DNA replicates before being digested.

So why does the difference in two “DNA fingerprints” produced by gel electrophoresis indicate that the DNA came from two different organisms? When restriction enzymes are used to cut DNA strands into pieces, the restriction sights are in different places due to the differences in the entire sequence of nucleotides in the DNA strand; therefore, no two pieces of DNA end up being cut into the same number, size, and abundance of fragments. These different sized pieces are referred to as restriction fragment length polymorphisms or RFLPs (often pronounced “riff lips”). The RFLPs are what are moving through the gel and producing the bands seen.

PURPOSE

In this experiment, you will analyze DNA samples (represented by colored dyes) using aspects of RFLP analysis such as gel electrophoresis.

SCENARIO

A DNA sample was collected at a crime scene. The police have two suspects in custody. A DNA sample was taken from each suspect. The DNA sample from the crime scene and the samples taken from the suspects were all digested with the same restriction enzyme. Due to the large size of the molecules, they were each split in half and run in two different lanes*. Therefore, the three samples occupy six lanes in the gel. In order for a “match” to be determined, BOTH lanes of suspect must match BOTH lanes of the crime scene sample.

*please note that this isn’t what is normally done and would be extremely difficult if not impossible to do; DNA samples are run as complete samples in a single lane; this “change” accommodates the limitations of using dyes instead of DNA

Prelab Questions: To be answered before the first day of lab.

Match the following terms used in biotechnology to the correct description of them.

_____ digest

_____ DNA fingerprinting

_____ gel electrophoresis

_____ PCR

_____ restriction endonuclease

_____ RFLP

_____ STR

group of techniques used to identify individuals by their DNA
the separation of DNA fragments from other parts of a sample by size
small, unique piece of DNA resulting from digestion
process that amplifies the amount of DNA obtained from a sample for testing
small section of DNA with a short repeat of nucleotides, such as CAGCAGCAGCAG
enzyme that cuts DNA at a specific site; probably obtained from a bacteria
term used to describe what is happening to the DNA when it is cut up by an enzyme

What technique is being used in this lab? ______

What is the product of this lab that will be analyzed called? ______

WHY are the products of DNA analysis called DNA fingerprints?

Why do the DNA fragments move toward the positive electrode?

Which move faster/farther, smaller fragments or larger fragments? ______Explain why.
What three pieces of information can we get from gel electrophoresis?

If one of the suspect’s DNA matches the DNA collected at the crime scene, does that prove his guilt? ______Explain.

What are some applications outside of crime scene analysis that DNA fingerprinting are important in?

SAFETY

Wear goggles and an apron
Use caution with electrical equipment
Wash hands thoroughly after cleaning up lab station

MATERIALS

Kit Contents:

QuickStrip Samples (stored in refrigerator)

Practice Gel Load Solution (stored in refrigerator)

UltraSpec-Agarose (3 g)

TAE Electrophoresis Buffer (50X)

1 3-mL graduated disposable pipette

10 small graduated disposable pipettes

Materials per Station:

1 7x15 prepoured agarose gel

1 gel electrophoresis chamber (Model #M6+)

1 power supply (could be shared with 2nd group at table)

1 pipette tip case with disposable pipette tips (could be shared with 2nd group at table)

1 35-µL fixed-volume pipette

1 strip Dye Samples (A-F) representing DNA

300 mL dilute TEA buffer (6 mL 50X + 294 mL distilled water)

PROCEDURE

DAY 1: Complete this section the day before you plan to load and run your gels.

Using a pipettor
The pipettor is off-white with a pink button and print on it.
Find the volume marking. It should say 35 µL.
The first thing you always do when using a pipettor is to put a tip on it.
Do not remove the tip from the case with your fingers.
Hold the pipettor in your hand with the finger-rest over the bottom joint of your index finger.
Holding on to the pipettor, place the end of it into the opening of a tip in the case and push it down into the tip firmly but slowly. Do not jam it on there.
Lift the pipettor away from the case. The tip should stay firmly attached.
Loading and unloading the pipettor:
Put your thumb on the pink button. Push down gently and note the first or “soft” stop when the button is about halfway depressed.
Continue pushing past the soft stop until the button is all the way down. This is the second or “hard” stop.
Practice feeling both the soft and hard stops until you are confident in your ability.

Push the button down to the SOFT stop.
Put the disposable tip into the solution you are loading.
Release the button.
Remove the pipettor with tip from the solution. You should see solution in the disposable tip. Solution should NEVER enter the actual pipettor.

UNLOADING

Insert the disposable tip connected to the pipettor into the well or microtube you are filling.
Push the button down to the HARD stop.
Remove the pipettor with tip from the well or microtube.
Release the button. If you do not remove the tip from the well or microtube before releasing the button, you will suck the solution back into the pipette.

PRACTICE

Practice loading and unloading the pipettor with a tip on it several times. When you are finished practicing, use your fingers to remove the disposable tip and throw it away.

DAY 2:

Loading your gel
Place your gel electrophoresis chamber on the white cardstock page near the purplepower supply box with the end with the black electrodes pointing away from you so that the red end is closest to you.
Place your casting tray with your gel on it in the electrophoresis chamber. The wells (little slits) go at the end with the black (negative) electrode. The ledge that sticks out on the tray fits into a slot right in front of the black electrode. Be careful not to tip the tray as your gel will slide off and most likely will break and be unusable.
Slowly, pour all of your electrophoresis buffer (all 300 mL) into the deep well at the end of the chamber with the red electrode. Allow the buffer to run across the top of the gel and into the deep well at the end of the chamber with the black electrode.
Look at the buffer level through the side of your chamber. Make sure that the entire surface of your gel is completely submerged and that the two deep wells are equal in depth. If either of these conditions is not met, notify your teacher before going on.
Place your strip of dye samples A through F in front of you. Sample A should go in the 1st well on the left, then B, C, D, E, and F in order moving toward the right.
Put a disposable tip on your pipettor.
Make sure the dye in sample well A is all at the bottom (all 6 wells should have equal volume; if they don’t, tap gently on the foil to make sure all liquid is at the bottom of the wells).
Carefully push the disposable tip of the pipettor through the foil and all the way down into the dye. Load 35 µL of the dye into the disposable tip. Review the LOADING section of the procedure if necessary.
Unload the dye into well A of your gel. Look from the side through the chamber wall at the tip going into the well if necessary. Remember, the light refracts as it enters the buffer and will make it difficult to judge where the pipette tip is. Once the tip is down in well A, unload the dye and remove the pipettor from the well BEFORE you release the pink button. Review the UNLOADING section of the procedure if necessary.

IMPORTANT:

Do not break through the bottom of the well or your dye will run into the buffer under the gel.
Be careful not to tear the well.
Throw away the disposable tip and put a new one on the pipettor before loading the next dye.

Repeat with dyes B through F. You should be able to see the dyes in all 6 wells. If you can not, tell your teacher before continuing.

Running your gel (electrophoresis)
Carefully place the lid on the electrophoresis chamber. You must line up the two electrodes correctly. DO NOT FORCE THE LID. If you can’t hold it level and slide it straight down with the black and red caps fitting over the metal prong inside, ask for help!
Make sure your power supply (purple box with the plug) is OFF and UNPLUGGED.
Plug the red wire into one of the red circles on the power supply and the black wire into the black circle directly to the left of the red circle.
Make sure the voltage toggle switch is in the center OFF position, THEN plug in the power supply to the wall.
If you are the only group using that power supply, turn the power supply on by moving the voltage toggle switch to 150V. If another group is sharing your power supply, do NOT plug it in or turn it on until AFTER the other group has everything plugged in.
Allow the current to run through your chamber for 10 minutes. At 10 minutes, look to see if you are beginning to see separation of the bands. If your bands have separated out between 1/3 and 1/2 the distance to the end of the gel for your gel or on BOTH gels (if two chambers are using same power supply), turn the power supply back to the off position and UNPLUG it. If you don’t see enough separation, allow the gel to run for another 5-10 minutes for a total of 15-20 minutes. Turn off the power supply and UNPLUG EVERYTHING.

IMPORTANT

Do NOT let your bands run off the end of the gel. If only one group’s gel has the bands getting close to the end of the gel, raise your hand and have your teacher look. If the bands run off the end of the gel you will NOT be able to analyze your data.

Once the power supply is OFF and everything is unplugged, remove the lid from the electrophoresis chamber.
Carefully lift out the casting tray with the gel still on it and place it on the lab table or on a white piece of paper to view your gel.

While you’re waiting:

Clean up everything you can at your lab station:
Throw away the parafilm (if applicable), ziplock baggie, tips, and strip that your dyes were in
When your gel is finished running and you’ve drawn it, throw the gel away, but NOT THE TRAY.
Rinse the tray and turn it upside down on a paper towel to air dry. Do NOT rub it dry with paper towels as you will scratch it.
Pour the buffer from the chamber into the sink, wash it down with plenty of water, rinse the base of the chamber only and turn it upside down on a paper towel to air dry. Do NOT rub it dry with paper towels as you will damage the delicate wires in the base of each deep well and will scratch it.

Prelab lecture discussion questions (to be answered at the end of Day 1):

Why is it important to use a new tip with each DNA (dye) sample? ______

Why is it important to remove the tip of the pipettor from the well or microtube before releasing the button on the pipettor? ______

At which end of the electrophoresis chamber do the wells go? ______

What is the purpose of the buffer going from the bottom of the deep well at one end of the chamber to the bottom of the deep well at the other end of the chamber and completely covering the gel? (What is it acting as?—think electricity and conduction) ______

______

Why is it important that your bands not run off the end of your gel? ______

Why is it important that the power supply is OFF before you plug it in? ______

______

DATA

The data from this lab is the results of your electrophoresis procedure. You should see a pattern of bands on your gel. The pattern & thickness of your bands is very important.

Using colored pencils, sketch the bands as they appear on your gel.

A B C D E F

ANALYSIS & CONCLUSION

Look at Figure 1 above and at your gel sketch. Look at the banding patterns and colors in Lanes A and B which were the DNA samples from the crime scene. Then compare the banding patterns and colors of lanes C and D to see if they match lanes A and B. Lanes C and D are DNA samples taken from Suspect 1. Then compare the banding patterns and colors of lanes E and F to see if they match lanes A and B. Lanes E and F had the DNA samples that were taken from Suspect 2.

Which suspect has both lanes that match the crime scene DNA lanes? ______

What does that mean for the suspect?______

If the crime scene is a burglary at a home and the suspect you identified in question 2 lives in that house, how does that impact your use of the word “suspect”? ______

What would have been the purpose of collecting the DNA of someone who lives in a house where a crime was committed? ______

What kind of evidence would you look for at a crime scene to obtain DNA? ______

Can you think of a case when two people will have identical DNA patterns? ______

If this lab used actual DNA instead of dyes, restriction enzyme would have been added to each sample before it was loaded into the gel. Answer these questions based on the concepts that would apply if this lab used digested DNA.

If there are 5 bands visible on the gel for one sample, how many restriction sites were there? _____ Explain. ______

As you look at the gel, you notice that some of the bands of DNA are thicker than others. What does that mean? ______