|Lab 6B – DNA Fingerprinting|
Restriction enzymes are endonucleases that actually cut the phosphodiester bonds on the sides of deoxyribonucleic acid. These endonucleases recognize specific DNA sequences in double-stranded DNA, which is usually a four to six base pair sequence of nucleotides. The endonucleases then digest the DNA at these sites. The resulting product is usually fragments of DNA of various lengths. Some restriction enzymes cut cleanly through the DNA double helix while some produce uneven or sticky ends. By using the same restriction enzyme to cut DNA from different organisms, the sticky ends produced will be complementary and the DNA from the two different sources can be recombined. In humans, no two individuals have the exact same restriction enzyme pattern in the DNA except for identical twins. In DNA, the antiparallel strands are difficult to deal with considering the restriction enzymes cut from opposite directions. This is the reason for the complementary ends. The restriction enzymes are named according to a system of nomenclature. The first letter represents the genus name of the organism. The next two letters come from the species name. If there is a fourth letter, it stands for the strain of the organism. Finally, if there are Roman numerals, it represents whether that particular enzyme was the first or second etc. isolated in that category.
In the electrophoresis chamber, there is placed an agar gel. This gel has wells in it for the samples of DNA to go into. The agarose gel is covered in a buffer so that the DNA is in a neutral pH solution. That way, the DNA moves in the direction its charge forces it. Since the phosphate groups on the skeleton of DNA are negatively charged, the whole molecule takes on the negative charge. So, when the DNA is placed inside the gel and the electricity turned on so that the poles are drawing the DNA toward the positive side, it will move through the gel and separate according to the size of the fragments.
By way of electrophoresis, the fragments of DNA of lambda can be separated by the traveling of the fragments through agar gel according to fragment size; DNA fingerprinting has occurred.
The materials needed for this lab are the following: an electrophoresis chamber, an agarose gel, lambda DNA digested with endonucleases, tracking dye, micropipette and tips, running buffer, and an electrical supply.
Prepare the agar gel for the electrophoresis by microwaving it for the suggested amount of time. When the gel has sufficiently hardened, place it in the chamber, pour the running buffer over the gel and add the DNA samples into the wells with a micropipette. Next, set the correct voltage and turn on the electricity. Allow this to run until the DNA is almost to the end of the gel, but do not let it run all the way out. Next, obtain the stain and a staining tray and let the gel set in the stain for a while. Next, put the gel into distilled water so that the stain can be taken out of the gel itself, leaving the DNA stained a royal blue. Look at and measure the gel over a light box, and put data into the data table.
|Actual base pairing sequence||Measured Distance (mm)|
|570(may not be detected)|
|125(may not be detected)|
|Measured Distance (mm)||Interpolated base pairs sequence||Actual base pair sequence|
|Band 2||14||11,000||5,148 or 5,973|
Discuss each of the following factors:
Voltage used. If a higher voltage had been used, the DNA would have moved faster through the agar gel, and slower if the voltage was low.
Running time. If allowed to run longer, the DNA would have eventually ended up into the running buffer, and lost to the experiment. If not allowed to run long enough, the bands could merge and be unclear for reading.
Amount of DNA. If more DNA had been used, the bands would have been darker because more of the fragments would have traveled the same distance in the gel. The bands would only have been more distinct and distinguishable.
Reversal of polarity. Had the polarity been reversed, the DNA would have been drawn the other way through the gel, and ended up in the running buffer.
Two small restriction fragments of nearly the same base-pair size appear as a single band, even when the sample is run to the very end of the gel. What could be dome to resolve the fragments? Why would it work? I would take the endonucleases needed to get the two fragment sizes and run an electrophoresis experiment just using those two sizes. It would probably work because these two fragments just by themselves can’t or shouldn’t stay together all the way to the end of the gel.
What is a plasmid? How are plasmids used in genetic engineering? Plasmids are small rings of DNA. They are used in genetic engineering because it is considerably easier to manipulate them into taking up preferred genes than it is to change the DNA sequence of the whole cell.
What are restriction enzymes? How do they work? What are recognition sites? These enzymes are endonucleases that cut the phosphodiether bonds of the DNA. They only cut at specific proteins, the recognition site.
What is the source of restriction enzymes? What is their function in nature? They occur naturally in prokaryotes and are used to cut up invading viral DNA that happens to get through the cell wall and plasma membrane of the bacteria.
Describe the function of electricity and the agarose gel in electrophoresis. The electricity is used to pull the DNA in a certain direction so that it will separate. The gel is helpful because it is like a freeze frame that allows the fingerprinting to be visualized. This could not be done in liquid or any solid.
If a restriction enzyme digest resulted in DNA fragments of the following sizes: 4000, 2500, 2000, and 400 base pairs, sketch the resulting separation by electrophoresis. Show starting point, positive and negative electrodes, and the resulting bonds.
What are the functions of the loading dye in electrophoresis? How can DNA be prepared for visualization? The dye allows the DNA to be more distinct so that accurate measurements can be made in determining the distance traveled and the amount of bands.
Use the graph prepared from the lab data to predict how far (in mm) a fragment of 8000 base pairs would migrate. A piece of DNA of that size would probably run about 17.5 millimeters.
How can a mutation that alters a recognition site be detected by gel electrophoresis? If you ran the normal and the mutant at the same time, you could see the change in the band that would be in a different place because it wouldn’t allow the DNA to be cut in that place.
There were not too many errors that could have occurred in this lab, but some of the few include the adding DNA to the agar gel. The person transferring had to have a steady hand and good eyes so that the gel wasn’t poked and the DNA made it into the chamber without problems. The wrong DNA samples were added to the wells, but the right ones were identified and later labeled correctly, out of order.
In conclusion, DNA fingerprinting, or electrophoresis is used to determine the size of the fragments that are cut by restriction enzymes. Restriction enzymes only cut at their specific protein recognition sites. This is useful because no two restriction enzymes code for exactly the same recognition site, allowing for a “fingerprint” like uniqueness that is only possible with one’s DNA. From the data collected in the electrophoresis experiment, other sizes of parts can be hypothesized by following the size of the base pair to the line of best fit drawn on the log sheet. This tells you about how many millimeters the base pair would probably go if allowed the same circumstances.