<P> Formation of recombinant DNA requires a cloning vector, a DNA molecule that replicates within a living cell . Vectors are generally derived from plasmids or viruses, and represent relatively small segments of DNA that contain necessary genetic signals for replication, as well as additional elements for convenience in inserting foreign DNA, identifying cells that contain recombinant DNA, and, where appropriate, expressing the foreign DNA . The choice of vector for molecular cloning depends on the choice of host organism, the size of the DNA to be cloned, and whether and how the foreign DNA is to be expressed . The DNA segments can be combined by using a variety of methods, such as restriction enzyme / ligase cloning or Gibson assembly . </P> <P> In standard cloning protocols, the cloning of any DNA fragment essentially involves seven steps: (1) Choice of host organism and cloning vector, (2) Preparation of vector DNA, (3) Preparation of DNA to be cloned, (4) Creation of recombinant DNA, (5) Introduction of recombinant DNA into the host organism, (6) Selection of organisms containing recombinant DNA, and (7) Screening for clones with desired DNA inserts and biological properties . These steps are described in some detail in a related article (molecular cloning). </P> <P> Following transplantation into the host organism, the foreign DNA contained within the recombinant DNA construct may or may not be expressed . That is, the DNA may simply be replicated without expression, or it may be transcribed and translated and a recombinant protein is produced . Generally speaking, expression of a foreign gene requires restructuring the gene to include sequences that are required for producing an mRNA molecule that can be used by the host's translational apparatus (e.g. promoter, translational initiation signal, and transcriptional terminator). Specific changes to the host organism may be made to improve expression of the ectopic gene . In addition, changes may be needed to the coding sequences as well, to optimize translation, make the protein soluble, direct the recombinant protein to the proper cellular or extracellular location, and stabilize the protein from degradation . </P> <P> In most cases, organisms containing recombinant DNA have apparently normal phenotypes . That is, their appearance, behavior and metabolism are usually unchanged, and the only way to demonstrate the presence of recombinant sequences is to examine the DNA itself, typically using a polymerase chain reaction (PCR) test . Significant exceptions exist, and are discussed below . </P>

Which two discoveries led to advancements in recombinant dna technology