History of Cloning Download image as a PDF Use NEBcloner to find the right products and protocols for each cloning step. Many cloning methodologies and, more recently, kits have been developed to simplify and standardize these processes.
These steps typically utilize enzymes, such as nucleases, phosphatases, kinases and/or ligases. As such, they have become the workhorse for many molecular methods, such as protein expression, gene expression studies, and functional analysis of biomolecules.ĭuring the cloning process, the ends of the DNA of interest and the vector have to be modified to make them compatible for joining through the action of a DNA ligase, recombinase, or in vivo DNA repair mechanism. Plasmid vectors allow the DNA of interest to be copied in large amounts and, often, provide the necessary control elements to be used to direct transcription and translation of the cloned DNA. By physically joining the DNA of interest to the plasmid vector through phosphodiester bonds, the DNA of interest becomes part of the new recombinant plasmid and is replicated by the host. The plasmid is a small, circular piece of DNA that is replicated within the host, and exists separately from the host’s chromosomal or genomic DNA. At the same time, a plasmid vector is prepared in linear form using restriction enzymes or PCR. A vector/plasmid backbone that contains all of the components for replication in the hostĭNA of interest, such as a gene, regulatory element(s), or operon, etc., is prepared for cloning by excising it out of the source DNA using restriction enzymes, copying it using the Polymerase Chain Reaction (PCR), or assembling it from individual oligonucleotides.The DNA fragment of interest to be replicated.A molecular cloning reaction is typically comprised of the following two components: Molecular cloning refers to the process by which recombinant DNA molecules are produced and transformed into a host organism, where they are replicated.
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