Agarose gel
Agarose Gel Electophoresis of DNA Topoisomers
Introduction
DNA can exist as different isomers that change the confirmation of the DNA’s structure. DNA can be in a linear confirmation this is a relaxed confirmation as the DNA can rotate about its axis unconstrained. It can also exist as a nicked circle this is also a relaxed confirmation as the DNA strands can again rotate freely with respect to one another. Covalently closed circular DNA or cccDNA exists as a supercoil this is because the covalent phosphodiester bonds of the backbone constrain the DNA leading to the formation of supercoils. This tension that develops from the supercoiling is a store of energy that is vital for many biological processes such as DNA replication. The aim of this procedure is to show that the different confirmation of DNA migrate at different rates through the gel when a current is applied. We will use this feature of DNA to show the effect the enzyme Topoisomers 1B has on DNA topology.
Method see flow diagram
Results and Discussion
We performed three reactions using three different types of DNA, M13 single stranded and cccDNA and puc118 cccDNA, we also carried out a BamHI and a topoisomerase digest on these DNA strands. In lanes T 1, 2 and E,1 ,2 we unfortunately had no definitive bands representing the cccM13 DNA for either of the digests, this could have been because we forgot to add the DNA or mix the reaction correctly, this does not seem an unlikely explanation as there are bands representing the other forms of DNA, it is possible that the DNA we used was degraded or not present in the correct concentration. What we should have seen when we carried out electrophoreses of undigested cccM13 DNA is two bands representing super coiled and relaxed close circular DNA. When digested with BamHI these bands should have become one band of relaxed linear double stranded DNA. This is because BamHI cleave the DNA at one site breaking the covalent linkage allowing the DNA to unravel
Introduction
DNA can exist as different isomers that change the confirmation of the DNA’s structure. DNA can be in a linear confirmation this is a relaxed confirmation as the DNA can rotate about its axis unconstrained. It can also exist as a nicked circle this is also a relaxed confirmation as the DNA strands can again rotate freely with respect to one another. Covalently closed circular DNA or cccDNA exists as a supercoil this is because the covalent phosphodiester bonds of the backbone constrain the DNA leading to the formation of supercoils. This tension that develops from the supercoiling is a store of energy that is vital for many biological processes such as DNA replication. The aim of this procedure is to show that the different confirmation of DNA migrate at different rates through the gel when a current is applied. We will use this feature of DNA to show the effect the enzyme Topoisomers 1B has on DNA topology.
Method see flow diagram
Results and Discussion
We performed three reactions using three different types of DNA, M13 single stranded and cccDNA and puc118 cccDNA, we also carried out a BamHI and a topoisomerase digest on these DNA strands. In lanes T 1, 2 and E,1 ,2 we unfortunately had no definitive bands representing the cccM13 DNA for either of the digests, this could have been because we forgot to add the DNA or mix the reaction correctly, this does not seem an unlikely explanation as there are bands representing the other forms of DNA, it is possible that the DNA we used was degraded or not present in the correct concentration. What we should have seen when we carried out electrophoreses of undigested cccM13 DNA is two bands representing super coiled and relaxed close circular DNA. When digested with BamHI these bands should have become one band of relaxed linear double stranded DNA. This is because BamHI cleave the DNA at one site breaking the covalent linkage allowing the DNA to unravel