Separation of DNA and RNA by Gel Filtration Chromatography and Electrophoresis
Biology I Ė 101-NYA-05
Instructor: Martin Chance
Experiment date: October 26, 2001
Submitted: November 16, 2001
CEGEP John Abbot College
The goal of this experiment was to separate DNA from RNA using first gel filtration chromatography and then gel electrophoresis to analyze the results of the chromatography. The results obtained showed that the chromatography was only partly effective in separating the DNA from the RNA. The electrophoresis was shown to be a much more effective method then the chromatography for separating DNA from RNA. Suggestions for making the chromatography more effective include lengthening the column and using the chromatography technique several times on the remaining contaminated samples after the first chromatography.
The technique of gel filtration chromatography is used to separate molecules of different sizes and shapes (JAC Biology NYA Laboratory Outlines, 2001). The goal of this experiment is to take a mixture containing plasmid DNA and degraded RNA fragments and to separate the two kinds of nucleic acids. The process of gel filtration involves letting the mixture of RNA and DNA be carried by an elution buffer through a filtration matrix contained in a glass column. The matrix consists of microscopic beads which are porous (JAC Biology NYA Laboratory Outlines, 2001). The larger, irregularly shaped molecules are unable to penetrate the pores of the beads and instead they tend to flow around the beads while smaller, compact (often spherical) molecules tend to get trapped in the pores for awhile before passing through the matrix (JAC Biology NYA Laboratory Outlines, 2001). Therefore, a heavier molecule will pass through the matrix faster then a lighter one. If the separation is successful, the first samples that have passed through the matrix should contain DNA, while later samples should contain RNA, since the plasmid DNA has a higher molecular mass then the degraded RNA.
In order to test the effectiveness of the gel filtration chromatography, another process called gel electrophoresis is used. Gel electrophoresis again separates molecules according to their size and shape, but also according to their charge (JAC Biology NYA Laboratory Outlines, 2001). The samples collected during the chromatography are placed into a gel bed and when a voltage is applied across the gel bed, the molecules will migrate towards the pole they are attracted to. In the case of DNA and RNA the charge-to-mass ratio on the two types of nucleic acids is not a factor, because there are always two negatively charged phosphate groups for each base pair charge (JAC Biology NYA Laboratory Outlines, 2001). Both types of nucleic acid will migrate towards the positive pole, however the RNA will move faster through the gel bed because it is smaller. This is because the thicket of polymer fibers in the gel impede longer fragments more then shorter ones (Campbell, 1999). Essentially, the longer molecules are in contact with more fibers then shorter ones and are slowed by the added friction of the extra fibers.
After letting the gel electrophoresis run for awhile, it should be possible to see how well the gel filtration chromatography separated the DNA and the RNA. The first few samples should contain only DNA, which should be represented by a band which hasnít moved very far through the gel bed. The later samples should contain the RNA, which should be represented by bands which have moved farther through the gel bed.
Materials and Methods:
Refer to JAC Biology NYA laboratory outlines considering the following changes.
-In the section "Fraction Collection," during step 7, 27 drops were collected
-In the section "Fraction Collection," during step 8, 4 drops were placed in sample tubes 1, 2, and 3. As well, 12 drops were placed in sample tubes 5-9.
-In the section "Agarose Gel Bed Preparation," during step 3, a 500ml Erlenmeyer flask was used.
Click here to view a scan of the results
Discussion and Conclusion:
The scan of our gel clearly shows the effectiveness of both the gel filtration chromatography and the gel electrophoresis. The results of the separation by the chromatography can be discussed by looking at which samples contained pure DNA, which contained purely RNA and which had a mixture. The first two lanes were empty, containing neither DNA nor RNA. This merely suggests that the DNA mixture had not flowed through the matrix when these samples were taken. The first two samples must have contained only the elution buffer that was used to pack the matrix. The third sample contained only DNA and it is the only such pure sample, while the fourth and fifth samples contained a mixture of DNA and RNA. Since the third sample was the only one to contain only DNA, it was only one where the DNA completely separated from the RNA fragments. Thus, only the in the case of the third sample was the technique of gel filtration chromatography completely effective. The samples that contained a mixture of DNA and RNA are interesting since they show that some of the DNA and RNA fragments passed through the matrix in the same amount of time. All of the DNA should have passed through the matrix before any of the RNA did since the DNA is heavier then the RNA (JAC Biology NYA Laboratory Outlines, 2001). The samples that contained a mixture must have contained the heaviest RNA fragments that were closest to the DNAís molecular weight. The difference between the heaviest RNA and the lightest DNA should still have been enough to separate them. The length of the column may explain the result that they were not separated completely. If a longer column was used, it probably would have slowed down the RNA more, while still letting the DNA flow through with little obstruction and thus given a better separation. However, the column used did give samples 6, 7, 8, and 9 contained only RNA. Since these samples contained only RNA it is clear that some of the RNA contaminants were completely removed from the DNA, so the samples which contain both RNA and DNA are at least more pure then the original mixture. Perhaps if the samples which still contained a mixture were run through the matrix again, the separation would continue to improve. Since the lightest RNA fragments would not occupy the pores in the matrix, the pores might be able to slow down some of the heavier RNA which got through as quickly as the DNA during the first pass through the matrix. The tenth sample represented the control of the experiment, since it contained the mixture which had not gone through the process of chromatography. The sample shows that the original mixture did contain both DNA and RNA.
The separation power of the electrophoresis was much stronger then that of the chromatography as predicted by JAC Biology NYA Laboratory Outlines, 2001. The scan of the gel shows that all of the DNA has traveled less towards the anode then the RNA fragments. The RNA appears in blobs roughly twice as far away from the starting point as the bands of DNA. This was expected since the RNA fragments are smaller then the pieces of DNA and the thicket of polymer fibers in the gel impede longer fragments more then shorter ones (Campbell, 1999). The effect of the gel electrophoresis is most clearly seen in the tenth sample, which contained the original mixture that had not gone through the chromatography. Even without the help of the chromatographyís separation effect, the DNA and RNA are still completely separate in the gel. After the electrophoresis was run, the sections containing DNA could have been cut out and the DNA diluted off the gel to obtain a pure sample of DNA.
Campbell, Neil A. 1999. Biology. Menlo Park, California: Addison Wesley Longman, Inc. pp. 1175.
2001. Biology NYA Ė Laboratory Outlines. Ste. Anne-de-Bellevue: John Abbott College Bookstore. p.9-1 Ė p.9-10.
Ethical Discussion Paper: Should Every Baby Born in Canada be DNA Fingerprinted?
There are several ethical reasons for and against DNA fingerprinting, the process whereby a babyís genetic makeup would become known and predictions about the future health of the child could be made. I personally feel that genetic tests should be optional, but not obligatory. Some people do not want to know the future, preferring to take life as it comes and not live in fear of the eventual day when their child starts to show symptoms of a genetic disease he or she has. On the other hand, some people would prefer to have advance notice of genetic problems so that they can be better prepared for the eventual onset of the disease. I donít think that the decision between these two options should be made by the government, but rather that parents should be able to make the choice for themselves.
Part of the reason I donít think DNA fingerprinting should be mandatory is because it could lead to discrimination. For example, insurance companies will not want to insure people who are likely to develop a genetic disease when they are older. It is also possible that employers will not want to hire people who are likely to get health benefit payments because of a genetic disease. Understandably, the insurance company, or a prospective employer, would want to have genetic information about people in order to save money for their company. However, I donít think that it is fair to discriminate because of genetic factors. I essentially feel this way because people are not in control of their genetic material, and I think it is very unfair to base decisions on something that people have no control over.
Another aspect of DNA fingerprinting is pre-natal testing. One of the main reasons that prenatal DNA fingerprinting is being pushed for by the government relates to the public cost of raising children with genetic diseases. Some people feel that children who will develop genetic diseases should not be born. While I understand why people might feel upset about having tax dollars go towards covering the health bills of a child with a genetic disease, I donít think that it is fair for society to tell two parents that they must abort their child because of a disease that the child will have. If two parents have the test and choose to abort the child, I have no problem with that. However, I do not think that it is acceptable for the parents to be pressured into aborting the child. I feel that an abortion is a personal choice which prospective parents should be able to make on their own terms.
I still feel that genetic tests should be available for those who want them though. For example, two parents who know they are carriers for a disease may want to know if their child will have the disease they carry. However, I feel that overall, the tests should not be mandatory since decisions based on the results of the tests are personal and I donít think that our society has the right to make such decisions for people.