Analysis of Supercoiled Plasmid DNA Lab Report Laboratory 2 BTEC 3P93

Tatiana Machado Rodrigues da Cunha Instrutor: Matilda

February 12, 2014

1-Introduction The DNA is a long double-helical structure that if extended would not fit into the cell (valid for eukaryotes and prokaryotes), and because of that the DNA is compacted with various loops and coils. The supercoiling phenomena is responsible for part of this compaction and is metaphoric compared to the coiled telephone cord, which has a tendency of to twist itself, as well as the two strands. The term super is used because the helical coiling is over and above that of the twisting of the two strands in the double helix. This topological concept changes the shape of the DNA molecule creating different topomeisomers and it is link to the transcription and replication initiation or inhibition. Plasmids circular DNA that have large application in molecular genetics, one of them is the recombinant DNA technology. In bacteria the supercoiling is more common due the DNA circular that is constrained by having no termini. Its normal helical structure is called relaxed state, but somehow when one of the ends is twisted and the other is fixed, the DNA will be underwinding or overwinding producing negative or positive supercoils, respectively. Naturally, the negative supercoils occurs in circular DNA. James White proved mathematically this phenomenon as: L = T + W. Where L, the linking number (also symbolized Lk), is the number of times that one DNA strand winds about the other and is invariant as long the two strands remain intact. The twist represented by T is the number of complete revolutions that one strands makes around the duplex axis. It is positive for right-handed duplex DNA and normally this would be the number of base pairs divided by 10.4 (or 10.5, which is the number of base pairs per turn of B-DNA). And the W is the writhing number that is the number of turns that the duplex axis makes about the superhelix axis.

Some method is needed to remove supercoiling, allowing DNA to relax. It is easy to remove supercoiling from a strand of DNA simply by twisting either of the free ends, but a cyclic DNA molecule does not have ends. The coiled phone cord presents a similar problem. While the handset is in the cradle is impossible to remove the supercoiling, however once the handset is picked up you can remove the supercoiling by turning the handset. Otherwise, the enzymes called topoisomerases are used to remove supercoiling or change the supercoiled state of DNA for replication and transcription demands. There are two classes of topoimerases: I and II. The type I is the one that can unwind the duplex DNA by cutting single-stranded (passing a loop of a second strand through the break, and then resealing the break). Some restriction enzymes can also undo the supercoils cleaving the chain and releasing the ends, which results in a linear molecule free of torsion. To analyzethe topoisomers is used the Agarose Gel Electrophoresis. The intercalation of ethidium between the base pairs locally untwists the double helix , which, since the linking number of the circle is constant, is accompanied by an equivalent increase in the writhing number. As the negatively coiled superhelix untwists, it becomes less compact and hence sediments more slowly. At one point the DNA circles have bound sufficient ethidium to become fully relaxed. As the ethidium concentration is further increased, the DNA supercoils in the opposite direction, yielding a positively coiled superhelix. Thus when a series of topoisomers are run on a gel, the relaxed molecules migrate most slowly, while both negatively and positively supercoiled molecules migrate faster. Othe types of intercalates agents can be used as well, for example, the chloroquine an antimalarial drug. Since supercoiling is a higher energy structural form of DNA it actually favours binding of intercalators since this unwinds and relaxes the supercoiled state.

In this laboratory experiment was isolated a plasmid from a bacterial culture (or use some recently prepared) and visualized its various topological forms by separating them using agarose gel electrophoresis.

2- Results and Discussion The procedures was not finished due to an error that occurred during the final steps of the plasmid isolation. Although, was provide an example of the agarose gel with the pKK223-3, plasmid from the DH5 E. coli cells. The gel picture below shows two lanes of the same plasmid, but treated in different ways in the lane 1 probably was treat with certain amount of chloroquine and the lane 2 was treat with less quantity. It is known that lower concentrations of chloroquine, more negatively supercoiled molecules migrate further through the gel and more relaxed molecules migrate slower. At a higher concentration this will be reversed, with more relaxed molecules migrating further. This intercalator converts highly negative supercoiled topoisomers into less supercoiled ones. In the other hand, for the positively supercoiled topoisomers is the opposite, they gain extra supercoils and migrates faster. So, the fully relax DNA in the gel is the first band that appears in lane 1 and 2 stronger and the fully supercoiled bands is the one in the final of the gel of the lane 2, because it runs faster. The gel showed that DNA that had been relaxed to varying degrees and then resealed reveals that 12 bands separate native from fully relaxed, W= 11. Considering that, the plasmid pKK2223-3 has 4584 bp, and the using 10.4 base pair per turn, the T0 would be around 440.7.

Figure 1 Agarose gel electrophoresis pattern of plasmid pKK223-3. Below is a picture of a gel run with circular DNA from the Plasmid pAT153. Lane 1 of the gel has three visible bands as seen by ethidium bromide staining: the native supercoiled pAT153 S1, supercoiled dimer S2 and "nicked" circular DNA N. Lane 2 is a sample of the same DNA incubated with a topoisomerase. a) What is "nicked" DNA? Why does "nicked" DNA (N) migrate more

slowly that the native supercoiled DNA? Be explicit it your rationale. A nick is an isolated break in one of the two strands keeping the circular form intact and is a sufficient to relax a supercoiled DNA. This is because the sugar phosphate chain opposite the nick is free to swivel about its backbone bonds .The nicked DNA (open circular DNA) migrate more slowly because compact DNA as normal supercoiled structure move faster than circular structure (relaxed). Supercoiled structure has smaller area and will move through holes in gel faster. Circular structure has bigger area and hence it experiences more frictional resistance moving through the gel.

Figure 2 Nicked DNA b) What are all the other bands in lane 2? Usually in the agarose gel electrophoresis pattern of a sample of chemically identical DNA molecules with different linking numbers results in a series of discrete bands. The molecules in a given band all have the same linking number and differ from those in adjacent bands. c) What is the minimum linking number observable from this gel? To determine the linking number we need to know the size of the plasmid and Plasmid pAT153 has 3658, according to the literature. So, using the formula Tw0= N/10.4 we have Tw0= 3658/10.4= 351.7 and was counting 13 band, which means W=13. Put it in the formula Lk = Tw + Wr , we have Lk= 351.7 -13 = 338.7. Thus the minimum linking number in the gel would be 338.7. d) What would the gel look like after a few hours of incubation? The tendency of the gel is to has less bands due the action of the topoisomerase in the circular DNA that is remove the supercoils, thus the DNA will fully relaxed running in the gel slowly forming a gel looking more with the S2 band of the Lane 1, but more strong.

Figure 3 Agarose gel electrophoresis pattern of plasmid pAT153 3-Conclusion With this lab session we were able to learn how isolate plasmids and how to prepare an agarose gel. Also we could understand more about the DNA strutucture and the enzymes related to it. The agarose gel electrophoresis method can be used to determine the supercoiled density of the circular DNA, but do not give enough information about the shape of these structures. For better analysis would be used the electron microscopy. 4-References Mirkin, S. M. (2001). DNA Topology: Fundamentals. Encyclopedia of Life Sciences. doi: 10.1038/npg.els.0001038 Van Vranken, D. L., & Weiss, G (2012). Introduction to bioorganic chemistry and chemical biology. New York, USA: Garland Science Title. Voet, D., & Voet, J. G. (2011). Biochemistry / Donald Voet, Judith G. Voet. Hoboken, NJ : John Wiley & Sons, c2011.

Watson, J. D., Baker, T. A., Bell, S. P., & Gann, A. (2013) Molecular Biology of the Gene (7th ed.). USA: Pearson.