Many types of bacteria contain plasmid DNA. Plasmids are extra-chromosomal, double-stranded circular DNA molecules generally containing 1,000 to 100,000 base pairs. Even the largest plasmids are considerably smaller than the chromosomal DNA of the bacterium, which can contain several million base pairs. Certain plasmids replicate independently of the chromosomal DNA and can be present in hundreds of copies per cell.
A wide variety of genes have been discovered in plasmids. Some of them code for antibiotic resistance and restriction enzymes. Plasmids are extremely important tools in molecular cloning because they are useful in propagating foreign genes. When plasmids are used for these purposes, they are referred to as vectors.
Through the use of recombinant DNA technology, hundreds of artificial vectors have been constructed from elements of naturally occurring plasmids. These vectors have specifically designed properties that make them useful in solving particular experimental problems. For example, synthetic oligodeoxynucleotide linkers have been incorporated into many plasmid vectors. These linkers contain many different restriction enzyme recognition sites to facilitate the insertion of foreign DNA. The linkers are often placed near characteristic marker genes or high efficiency transcriptional promoters, both of which aid in the isolation and expression of the cloned DNA.
Plasmid DNA naturally exists as a supercoiled molecule. Supercoiling arises from alterations in the winding of the two DNA strands around each other. In certain areas of the molecule, the DNA strands are wound around each other less frequently than in non-supercoiled DNA. The strain caused by these alterations create deformations in the DNA. These deformations partially relieve the strain and ultimately lead to supercoiling. Supercoiled DNA is folded onto itself and has a more condensed and entangled structure than the same DNA which is relaxed. As an analogy to supercoiling, consider a rubber band. When the rubber band is twisted, it eventually becomes knotted and collapses onto itself as an entangled ball.
Purified DNA must be a covalently closed circle to exist as a supercoiled molecule. Supercoiled plasmid DNA is often called Form I DNA. Supercoiling in the cell is caused by the action of enzymes called DNA gyrases. These enzymes use the chemical energy in ATP to introduce supercoiling into a relaxed molecule. In addition, there are enzymes that relax supercoiled DNA and are called unwinding or relaxing enzymes. Supercoiling has important biological consequences. Very large DNA molecules would simply not fit in the cell if they were not supercoiled. Gene expression can also be influenced by supercoiling.
If one or more phosphate bonds anywhere in the backbone of supercoiled DNA are broken, the molecule unravels to a relaxed form called open circular DNA or Form II DNA. These breaks in the phosphate backbone are called nicks. Nicked double-stranded DNA is not covalently closed.
The two strands of nicked DNA are still held together by hydrogen bonds between the bases. With time, purified supercoiled DNA slowly develops nicks and converts to Form II. This is because supercoiled DNA is not as stable as its relaxed or open circular forms. Endonucleases, such as DNAse I, will randomly nick supercoiled DNA when used in low amounts. Nicking can also be introduced by mechanical manipulations during plasmid purification.
During replication, several of the same plasmid molecules can form interlocked rings. These multimers of plasmid are called catenanes. A catenane containing two of the same plasmid molecules is called a dimer. Similarly, those containing three or four molecules are called trimers and tetramers, respectively. Each plasmid molecule in a catenane can be supercoiled, however, for clarity, they are represented as relaxed circles.
Agarose gel electrophoresis is a powerful separation method frequently used to analyze plasmid DNA. The agarose gel consists of microscopic pores that act as a molecular sieve. Samples of DNA can be loaded into wells made in the gel during molding. When an electric field is applied, the DNA molecules are separated by the pores in the gel according to their size and shape. Generally, smaller molecules pass through the pores more easily than larger ones. Since DNA has a strong negative charge at neutral pH, it will migrate towards the positive electrode in the electrophoresis apparatus. The rate at which a given DNA molecule migrates through the gel depends not only on its size and
shape, but also on the type of electrophoresis buffer, the gel concentration and the applied voltage. Under the conditions that will be used for this experiment, the different forms of the same plasmid DNA molecule have the following rates of migration (in decreasing order):
Supercoiled > linear > Nicked Circles >dimer > trimer > etc.
Supercoiled DNA has the fastest migration rate of the different forms of plasmid. In the plasmid extraction experiment you will be doing, there will be some residual, degraded RNA which consists of transfer RNA and digested ribosomal and messenger RNA. Degraded RNA has a faster migration rate than supercoiled plasmid DNA because it is much smaller in size.
In the first step of the experiment a cell lysis solution is added to the cells. This solution contains the detergent sodium dodecyl sulfate (SDS) which dissolves the cell membrane and denatures proteins. The solution is very alkaline (pH > 12) due to the presence of sodium hydroxide. The high pH aids in denaturing proteins and causes the cleavage of the phosphate bonds in RNA. This eliminates interference from high molecular weight RNA during the plasmid purification. Under highly alkaline conditions, the two strands in non-supercoiled DNA (linear fragments of chromosomal DNA, relaxed and nicked circular DNA) separate and are partially removed from solution. However, this does not occur with supercoiled forms of plasmid DNA because the two strands are intertwined and entangled in a way that prevents them from coming apart. Therefore, supercoiled plasmid remains free in solution.
The potassium acetate neutralization buffer contains acetic acid and potassium salts. The acidic buffer neutralizes the alkaline conditions created by the sodium hydroxide. The potassium causes the SDS, with its associated membrane fragments and proteins, to precipitate. The chromosomal DNA of E. coli is attached at several points to the cell membrane. Centrifugation of the potassium-SDS-membrane complexes also removes large amounts of entrapped chromosomal DNA.
The addition of isopropanol precipitates the plasmid and remaining RNA. Tris buffer (diluted buffer concentrate for RNase) is used to resuspend the DNA precipitate in a higher concentration. The buffer contains the enzyme RNase, which further degrades RNA. The concentrated gel loading solution prepares the sample for electrophoresis by making it denser than the electrophoresis buffer. This enables the sample to sink into the wells of the submerged gel. A negatively charged, blue tracking dye is also present to monitor the electrophoresis and to make sample loading easier.
In this experiment, a 3000 base pair plasmid will be extracted from E. coli cells. The restriction map for this plasmid has a single site for Eco RI. Digestion with the enzyme will yield a single band measuring 3,000± 300 nucleotides. The multiple forms of the plasmid will be converted to the linear form.