Molecular cloning

Molecular cloning is a set of experimental methods in molecular biology that are used to assemble recombinant DNA molecules and to direct their replication within host organisms. The use of the word cloning refers to the fact that the method involves the replication of one molecule to produce a population of cells with identical DNA molecules. Molecular cloning generally uses DNA sequences from two different organisms: the species that is the source of the DNA to be cloned, and the species that will serve as the living host for replication of the recombinant DNA. Molecular cloning methods are central to many contemporary areas of modern biology and medicine. Molecular cloning is a set of experimental methods in molecular biology that are used to assemble recombinant DNA molecules and to direct their replication within host organisms. The use of the word cloning refers to the fact that the method involves the replication of one molecule to produce a population of cells with identical DNA molecules. Molecular cloning generally uses DNA sequences from two different organisms: the species that is the source of the DNA to be cloned, and the species that will serve as the living host for replication of the recombinant DNA. Molecular cloning methods are central to many contemporary areas of modern biology and medicine. In a conventional molecular cloning experiment, the DNA to be cloned is obtained from an organism of interest, then treated with enzymes in the test tube to generate smaller DNA fragments. Subsequently, these fragments are then combined with vector DNA to generate recombinant DNA molecules. The recombinant DNA is then introduced into a host organism (typically an easy-to-grow, benign, laboratory strain of E. coli bacteria). This will generate a population of organisms in which recombinant DNA molecules are replicated along with the host DNA. Because they contain foreign DNA fragments, these are transgenic or genetically modified microorganisms (GMO). This process takes advantage of the fact that a single bacterial cell can be induced to take up and replicate a single recombinant DNA molecule. This single cell can then be expanded exponentially to generate a large amount of bacteria, each of which contain copies of the original recombinant molecule. Thus, both the resulting bacterial population, and the recombinant DNA molecule, are commonly referred to as 'clones'. Strictly speaking, recombinant DNA refers to DNA molecules, while molecular cloning refers to the experimental methods used to assemble them. The idea arose that different DNA sequences could be inserted into a plasmid and that these foreign sequences would be carried into bacteria and digested as part of the plasmid. That is, these plasmids could serve as cloning vectors to carry genes. Virtually any DNA sequence can be cloned and amplified, but there are some factors that might limit the success of the process. Examples of the DNA sequences that are difficult to clone are inverted repeats, origins of replication, centromeres and telomeres. Another characteristic that limits chances of success is large size of DNA sequence. Inserts larger than 10kbp have very limited success, but bacteriophages such as bacteriophage λ can be modified to successfully insert a sequence up to 40 kbp. Prior to the 1970s, the understanding of genetics and molecular biology was severely hampered by an inability to isolate and study individual genes from complex organisms. This changed dramatically with the advent of molecular cloning methods. Microbiologists, seeking to understand the molecular mechanisms through which bacteria restricted the growth of bacteriophage, isolated restriction endonucleases, enzymes that could cleave DNA molecules only when specific DNA sequences were encountered. They showed that restriction enzymes cleaved chromosome-length DNA molecules at specific locations, and that specific sections of the larger molecule could be purified by size fractionation. Using a second enzyme, DNA ligase, fragments generated by restriction enzymes could be joined in new combinations, termed recombinant DNA. By recombining DNA segments of interest with vector DNA, such as bacteriophage or plasmids, which naturally replicate inside bacteria, large quantities of purified recombinant DNA molecules could be produced in bacterial cultures. The first recombinant DNA molecules were generated and studied in 1972. Molecular cloning takes advantage of the fact that the chemical structure of DNA is fundamentally the same in all living organisms. Therefore, if any segment of DNA from any organism is inserted into a DNA segment containing the molecular sequences required for DNA replication, and the resulting recombinant DNA is introduced into the organism from which the replication sequences were obtained, then the foreign DNA will be replicated along with the host cell's DNA in the transgenic organism. Molecular cloning is similar to polymerase chain reaction (PCR) in that it permits the replication of DNA sequence. The fundamental difference between the two methods is that molecular cloning involves replication of the DNA in a living microorganism, while PCR replicates DNA in an in vitro solution, free of living cells.