Using a second enzyme, DNA ligase, fragments generated by restriction enzymes could be joined in new combinations, termed recombinant DNA. 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. 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. This changed dramatically with the advent of molecular cloning methods. 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. 5.1 Genome organization and gene expression.4.7 Screening for clones with desired DNA inserts and biological properties.4.6 Selection of organisms containing vector sequences.4.5 Introduction of recombinant DNA into host organism.4.4 Creation of recombinant DNA with DNA ligase.4.1 Choice of host organism and cloning vector.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. There is also a lower chance of success when inserting large-sized DNA sequences. Examples of the DNA sequences that are difficult to clone are inverted repeats, origins of replication, centromeres and telomeres. Virtually any DNA sequence can be cloned and amplified, but there are some factors that might limit the success of the process. That is, these plasmids could serve as cloning vectors to carry genes. 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. Strictly speaking, recombinant DNA refers to DNA molecules, while molecular cloning refers to the experimental methods used to assemble them. Thus, both the resulting bacterial population, and the recombinant DNA molecule, are commonly referred to as 'clones'. 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. 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. Because they contain foreign DNA fragments, these are transgenic or genetically modified microorganisms ( GMO). This will generate a population of organisms in which recombinant DNA molecules are replicated along with the host DNA. The recombinant DNA is then introduced into a host organism (typically an easy-to-grow, benign, laboratory strain of E. Subsequently, these fragments are then combined with vector DNA to generate recombinant DNA molecules. 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. Molecular cloning methods are central to many contemporary areas of modern biology and medicine. 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. 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 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. Diagram of molecular cloning using bacteria and plasmids.

Pjet vector in serial cloner