DNA Fingerprinting (RFLP Analysis)
Introduction
DNA fingerprinting is a technique that is used to identify patterns that occur in DNA. No two organisms have identical DNA so this procedure can be used to identify if a sample of DNA came from a particular individual.
We will use this procedure in lab to identify whether a sample of DNA found at a crime scene belongs to one of three suspects.
The technique has a variety of other uses that will be discussed in lecture class. For example, it can be used to identify whether individuals carry genes for certain genetic diseases.
Restriction Enzymes
The technique of DNA fingerprinting requires that the DNA be cut up into small fragments. Restriction enzymes are used to perform this digestion.
Restriction enzymes were discovered in bacteria, which use them as a defense mechanism to cut up the DNA of viruses or other bacteria.
Hundreds of different restriction enzymes have been isolated. Each one cuts DNA at a specific base sequence. For example, EcoRI always cuts DNA at GAATTC as indicated below.
The sequence GAATTC appears three times in the DNA strand below. As a result, the strand is cut into four pieces.
Other restriction enzymes cut at different sites, some examples are listed below.
Enzyme Cutting Site Bam HI GGATCC Hae III GGCC Pst I CTGCAG Hinf I GANTC
In RFLP analysis, the DNA of an organism is cut up into fragments using restriction enzymes. A large number of short fragments of DNA will be produced.
Restriction enzymes always cut at the same base sequence. Because no two individuals have identical DNA, no two individuals will have the same length fragments. For example, the enzyme EcoRI always cuts DNA at the sequence GAATTC. Different people are going to have different numbers of this particular sequence and will therefore have different fragment lengths. In addition, some of them will be at different locations on the chromosome.
Gel Electrophoresis
Electrophoresis is a technique used to separate the DNA fragments according to their size. They are placed on a sheet of gelatin and an electric current is applied to the sheet. DNA is charged and will move in an electric field toward the positive pole.
In the diagram below, holes (wells) in the gelatin can be seen. DNA samples placed in these wells will migrate through the gelatin toward the + side after an electric current is applied.
The smallest fragments will move the fastest because they are able to move through the pores in the gelatin faster. Bands will be produced on the gelatin where the fragments accumulate. The shortest fragments will accumulate near one end of the gelatin and the longer, slower-moving ones will remain near the other end.
In the diagram below, four samples of DNA were placed on the gelatin. After an electric current was applied for a period of time, the fragments separated. Notice that sample D on the right does not match the other three samples.
The DNA bands must be stained to make them visible. Ethidium bromide-stained DNA will fluoresce when illuminated with UV light.
PCR techniques are used to produce sufficient quantities of DNA for this technique.
Procedure - Day 1
Setting Up the Apparatus
DNA fragments will be separated on agarose gels that have been prepared prior to today's lab. The table below shows the samples that will be used.
Component (tube) Origin of DNA Enzyme used to cut the DNA A Crime scene 1 B Crime scene 2 C Suspect 1 1 D Suspect 1 2 E Suspect 2 1 F Suspect 2 2 1. Remove the tape from the end of the gel trays if it has not been done already.
2. A plastic comb was used to create the wells for the samples. Carefully remove this comb.
3. Place the gel tray in the electrophoresis apparatus. The wells should be placed nearest the negative (black) electrode.
4. Add enough buffer solution so that the gel is completely submerged.
5. The gel has eight lanes but you will need six for your samples. The two outside lanes can be used to practice loading samples in the wells. Use the practice loading solution for this purpose.
6. Load 25 ul of sample A into the second well from the left. Repeat this procedure, placing each of the remaining samples (B through F) in a different well. Record what samples that you placed in each of the wells. This can be done by writing the sample letter and the well number for that sample.
Connecting the Power Supply
7. Place the lid on the apparatus. The red and black electrodes on the base should match the electrode connections on the lid.
8. Connect the apparatus to the transformer. This transformer can be used to run two different gels.
9. Switch on the transformer using the switch on the right side near the back.
10. Use the button to the right of the LED display on the transformer to select "V."
11. Use the up and down arrows to the left of the LED display to adjust the voltage to 125.
12. To start power flow to the gel, press the button on the right side of the front panel. This button shows a drawing of a person running. The green light next to this button indicates that the power is on. Check for the production of bubbles in the electrophoresis apparatus to confirm that the power is turned on.
Running the Gels
13. The gels should run for about 35 minutes. Each sample contains a marker dye that runs just ahead of the smallest DNA fragments. The electricity should be switched off when this dye approaches the end of the gel. Do not let the dye run off of the gel.
14. When the gels have finished running, switch off the power, disconnect the apparatus, and remove the lid.
Staining the Gels
15. Remove the plastic gel holder and slide the gel into a plastic staining tray. Do not attempt to pick up the gel with your hands; it is fragile and will break.
16. Place a blue staining sheet on the gel so that the blue surface contacts the gel.
17. Slide your fingers over the entire surface of the staining paper several times to promote the transfer of stain from the paper to the gel.
If you are doing this laboratory exercise in one day, proceed to step 18 below. If you are doing it in 2 days (staining overnight) go to step 19.
18. Place the gel holder on top of the staining paper as shown in the photograph below. This will ensure that the paper makes contact with the gel. Allow the gel, staining paper, and plastic gel holder to remain in place for ten minutes to transfer the dye to the gel. Go to step 21 (day 2) below and continue.
19. Place the gel holder on top of the staining paper as shown in the photograph below. If you are doing this exercise in 2 lab periods, allow the gel, staining paper, and plastic gel holder to remain in place for several minutes to ensure that the paper makes contact with the surface of the gel. Next, remove the gel holder but keep the staining paper in contact with the gel. Empty any water in the tray and then wrap the tray with the gel and staining paper in plastic wrap so that it remains moist until the next lab period. Your instructor will place the gel in a refrigerator.
Procedure - Day 2
Destaining the Gels
20. Unwrap your gel and place it in a plastic staining tray.
21. In order to see the DNA, you must rinse the stain from the gel (called destaining). Unwrap the tray with the gel prepared in the previous class. Remove the staining paper and discard it in the trash.
22. Add warm water (approximately 37 degrees) to the tray containing the gel and allow this to sit for 5 to 10 minutes. You should occasionally stir or agitate the tray during this time to promote destaining. Empty the water and repeat this destaining procedure one or two more times until the DNA is clearly visible.