Plasmid Isolation and Restriction Enzyme Mapping
Introduction
Plasmids are circular, double stranded extrachromosomal DNA molecules that are found in bacteria which can self-replicate. They are naturally occurring DNA molecules advantageous to the host bacterium by carrying genes which specify metabolic capacities. (Garrett et al., 2010) Besides, plasmids exist in a wide variety of sizes from a few thousands to hundreds of thousands of base pairs. Many plasmids have been engineered to serve as plasmids cloning vectors to carry genes. (Synder et al., 2007) Useful plasmids that serve as cloning vectors have three common features which are an origin of replication, a selectable marker and a cloning site. (Garrett et al., 2010) Plasmid pBR322 is an example of one of the widely used cloning vector that has 4363 base pairs. This plasmid has an origin of replication, a selectable marker which codes for ampicillin and tetracycline resistance gene, and a multiple cloning site.
The structure of plasmids is mostly circular with no ends, except with a few known plasmids which are linear. Plasmids can also be supercoiled in structure when the strands are prevented from separating and there are no ends to rotate (Synder et al., 2007). Three conformations that plasmid DNA can exist in are supercoiled, open-circular (nicked), and linear. Majority of plasmid DNA is supercoiled after plasmid preparation, with a proportion of it sustaining nicks (Varsani et al., 2013). The plasmid remains circular when there is a nick which results in a break in only one strand of a duplex. However, the break allows DNA to rotate around the phosphodiester backbone which causes supercoils to be released. Supercoiled plasmid is more compact and sustains less resistance compared to open circular plasmid and therefore travels quicker in an agarose gel. Linear conformation is produced when a smaller fraction of the DNA sustains double-stranded breaks and it has less resistance than open-circular DNA, but more resistance than supercoiled DNA (Varsani
Plasmids are circular, double stranded extrachromosomal DNA molecules that are found in bacteria which can self-replicate. They are naturally occurring DNA molecules advantageous to the host bacterium by carrying genes which specify metabolic capacities. (Garrett et al., 2010) Besides, plasmids exist in a wide variety of sizes from a few thousands to hundreds of thousands of base pairs. Many plasmids have been engineered to serve as plasmids cloning vectors to carry genes. (Synder et al., 2007) Useful plasmids that serve as cloning vectors have three common features which are an origin of replication, a selectable marker and a cloning site. (Garrett et al., 2010) Plasmid pBR322 is an example of one of the widely used cloning vector that has 4363 base pairs. This plasmid has an origin of replication, a selectable marker which codes for ampicillin and tetracycline resistance gene, and a multiple cloning site.
The structure of plasmids is mostly circular with no ends, except with a few known plasmids which are linear. Plasmids can also be supercoiled in structure when the strands are prevented from separating and there are no ends to rotate (Synder et al., 2007). Three conformations that plasmid DNA can exist in are supercoiled, open-circular (nicked), and linear. Majority of plasmid DNA is supercoiled after plasmid preparation, with a proportion of it sustaining nicks (Varsani et al., 2013). The plasmid remains circular when there is a nick which results in a break in only one strand of a duplex. However, the break allows DNA to rotate around the phosphodiester backbone which causes supercoils to be released. Supercoiled plasmid is more compact and sustains less resistance compared to open circular plasmid and therefore travels quicker in an agarose gel. Linear conformation is produced when a smaller fraction of the DNA sustains double-stranded breaks and it has less resistance than open-circular DNA, but more resistance than supercoiled DNA (Varsani
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