- A restriction endonuclease “cuts” two DNA molecules at the same location. What can you assume is identical about the molecules at that location?
The DNA molecules must have identical palindromes that the enzyme cuts at the restriction site.
What are restriction enzymes? How do they work? What are restriction sites?
Restriction enzymes are degradative enzymes that recognize certain palindromes and cuts up DNA that is foreign to a bacterium. The enzymes cut covalent phosphodiester bonds of both strands at the restriction sites.
- What is the source of restriction enzymes? What is their function in nature?
Restriction enzymes occur naturally in prokaryotes and are used to cut up invading viral DNA that happened to get through the cell wall and plasma membrane of the bacteria by being injected into them by bacteriaphage.
- Describe the function of the following in gel electrophoresis:
- Electricity
The electricity creates a current, which pulls the negatively charged DNA toward the positively charged agarose gel so that it will separate.
- Agarose gel
The Agarose gel acts as a molecular sieve through which different sized DNA fragments travel and eventually separate into bands of similar sized fragments. It also allows the fingerprinting to be visualized.
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Discuss how each of the following errors would affect the results of electrophoresis:
- Keeping the electricity on longer than it is needed
If the electricity was kept on longer than necessary, the DNA would have eventually ended up into the running buffer and the experiment would be lost. If not allowed to run long enough, the bands could merge and be unclear for reading.
- Not using enough DNA in your sample
If the DNA used was not a sufficient amount, the bands would be very hard to see because they would be very thin. 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.
- Reversing polarity (switching the negative and positive ends of the gel)
If the polarity were reversed, the DNA would have been drawn the other way through the gel and ended up in the running buffer
- Forgetting to add the restriction enzymes to the DNA.
If there were no restriction enzymes added to the DNA, it would not have separated into different bands and all the DNA would have its original length and move together through the gel.
- Our lab was a simulation of an actual forensic case. If we had profiled real human DNA, what additional steps would we need in our procedure and why? Describe in detail.
Restriction fragment preparation and electrophoresis occur as they normally would. The third step would be blotting, which is when the capillary action pulls alkaline solution upward through the gel and through a nitrocellulose paper laid on top of it. The DNA is transferred to the paper, denaturing it in the process. The single strands of DNA would stick to the paper, positioned in bands exactly as it was on the gel. Hybridization with radioactive probes must occur next. The paper blot should be exposed to a solution containing radioactively labeled probe, which is single stranded DNA complementary to the DNA sequence. It attaches by base pairing to restriction fragments of complementary sequence. A sheet of photographic film is then laid over the paper in a process called Autoradiography. The radioactivity in the bound probe is exposes the film to form and image corresponding to specific DNA bands, which contain the DNA base paired with the probe.
- 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 done to resolve the fragments, if you could run the experiment again with the same DNA and restriction enzymes, but different equipment? Why would it work?
I would keep the electricity on longer so the DNA could eventually separate.
- The diagram below shows a segment of DNA with a total length of 4, 900 base pairs. The arrows indicate reaction sites for two restriction enzymes ( enzyme X and enzyme Y)
- Describe the results you would expect from the electrophoretic separation of fragments from the following treatments of the DNA segment above. Assume that the digestions occurred under appropriate condition and went to completion. Be sure to include the # of fragments produced, and the sizes of each fragment.
- DNA digested with only enzyme X
This DNA would contain 4 fragments, with lengths of 400, 1300, 1500, and 1700 bases.
- DNA digested with only enzyme Y
This DNA would contain 2 fragments, with lengths of 900 and 4000 bases.
- DNA digested with enzyme X and enzyme Y combined
This DNA would contain 6 fragments, with lengths of 400, 500, 1200, 1300, and 1500 bases.
- Undigested DNA
This DNA would be one fragment that would be 4900 bases long.
- Explain the different results you would expect if a mutation occurred at the recognition site for enzyme Y.
If there was a mutation at the recognition site for enzyme Y, the restriction enzyme would not cut the DNA and there would not be pieces of DNA cut from the enzyme Y.
Conclusion:
Restriction nucleases are important for recombinant DNA technology because they cut DNA at specific sites. These enzymes are usually made by bacterial species in which they degrade invading foreign DNA within the bacterial cell. Most restriction enzymes recognize a specific sequence of nucleotides in DNA and cut a long DNA double helix into restriction fragments, which are measured in the process of agarose gel electrophoresis.
Electrophoresis is the movement of charged particles in solution under the influence of an electric field. In gel electrophoresis, agarose gel is the stabilizing medium that serves as a matrix for the buffer in which the sample molecules travel. The gel is submerged in buffer within the electrophoretic gel cell. The samples are loaded into the sample wells in the gel, and electric current is passed through the gel.
Molecules of DNA are negatively charged because of negative charges on the phosphate group. In this exercise, nucleic acids migrate through the pores of the gel from the negative end of towards the positive end. The large DNA molecules move more slowly than smaller molecules, therefore molecules are sorted according to size.
