Another batch of DNA was treated in a likewise manner, except that nucleotides containing thymine were added instead of adenine. (Avril, 187-94)
When these two samples of DNA were mixed, the complementary "tails" of A- and T-bearing nucleotides became joined by hydrogen bonding. This combined the once separate fragments into long, interconnected chains. DNA ligase was then added to form bonds between the sugar and phosphate groups. The two DNA strands were now one.
It was certainly intriguing that one could now cut up DNA into unpredictable heterogeneous fragments and randomly stitch them back together. However, for further insights into the organization of DNA and its genes -- that is, the determination of precise nucleotide sequencesvery specific nucleases would have to be found. The prevailing opinion was that such specific DNA-cutting capability did not exist in nature.
The only clue to the possibility that more specific nucleases might exist came from observations beginning as early as 1953 that when DNA molecules from E. coli were introduced into another slightly different form of E. coli they seldom functioned genetically. They were quickly broken down into smaller fragments. This apparently was part of a system that had evolved in bacteria to protect them against the entrance of foreign DNA. In addition to all of the other more obvious forms of competition in nature, there is a constant invisible struggle played out in the microscopic world, in this case between bacteria and bacteriophages. Darwin's natural selection is recreated here on a minute scale. (David, 131-44)
First, bacteria can be grown under controlled conditions, rapidly and in enormous numbers. Overnight, a few cells will multiply into literally billions. It is very important to understand that a bacterial cell ordinarily reproduces simply by copying itself. Assuming that no mutations occur in the cells, all the descendants of that one cell are identical. Such a population of cells originating from a single cell is termed a "clone" and the process of producing that clone is referred to as "cloning" the cell.
The DNA in a typical bacterial cell exists in two forms. One is the single bacterial chromosome which, unlike the chromosomes in our cells, is in the form of a circular molecule. The DNA of all other organisms can be likened to a long string. In bacteria, the ends of the string are joined, forming a circle. In addition to the DNA in the bacterial chromosome, DNA also occurs in bacteria in the form of plasmids. These, like the bacterial chromosome, are also circular DNA molecules, but much smaller. When the bacterial cell divides, the bacterial chromosome replicates and one chromosome is passed on to the new cell. Likewise, each of the plasmids replicate and half are delivered to the next generation. The plasmids are unique, independent, self-replicating DNA molecules which can exist only within the living bacterial cell.
Plasmids can easily be isolated from bacteria by breaking open the cells with enzymes which break down the cell wall. The resulting mix is centrifuged.The heavier chromosomal DNA, termed "genomic" DNA, as well as cell fragments will go to the