Plasmid vectors are the simplest cloning vectors. It is a double stranded DNA molecule, can able to replicate autonomously. ORI is a specific sequence of nucleotide in DNA from where replication starts. Cloning vectors contains a cloning site also known as restriction site, to insert foreign DNA, cut by a restriction enzyme. Copy number is an essential feature of an ideal vector. Herbert Boyer, Keiichi Itakura, and Arthur Riggs were three scientists working in the Boyer’s lab, University of California, where they recognized a general cloning vector. Infection by λ phage requires adsorption of tail fibers on the cell surface, contraction of the tail, and injection of the DNA inside the cell. Standard methods of transformation are inefficient. Any DNA molecule that has the ability to replicate inside the host to which the desired gene is integrated for cloning- include plasmids, bacteriophages, cosmids, BAC, yeast vectors, shuttle vectors etc. 322= number of plasmid discovered in the same lab. 2. A vector is a DNA molecule which is used for transporting exogenous DNA into the host cell. These protein-encoding genes are located near the ori. Molecular gene cloning is difficult without the use of the cloning vectors. Figure: Schematic representation of the pBR322 plasmid, one of the first plasmids widely used as a cloning vector. It possesses the yeast telomere at each end. it avoids the possibility of insertional mutagenesis. The vector therefore contains features that allow for the convenient insertion or removal of a DNA fragment to or from the vector, for example by treating the vector and the foreign DNA with a restriction enzymethat cuts the DNA. They are utilized for gene transfer or gene delivery into human cells. Characteristics of a Cloning Vector Origin of replication (ORI) This process marks autonomous replication in vector. It is most widely used for gene cloning. Also, they can carry up to 45 kb of insert compared to 25 kb carried by plasmids and λ. Cosmids cannot accept more than 50 kb of the insert. Features of cloning vector. Both yeast and bacterial cells can be used as hosts. The molecular analysis of DNA has been made possible only after the discovery of vectors. They are capable of incorporating the bacteriophage λ DNA segment. Plasmid vector. CLONING VECTOR. Cloning vector is used for replicating donor DNA fragment within host cell. Cloning Vectors. CLONING VECTORS •Cloning vectors are DNA molecules that are used to "transport" cloned sequences between biological hosts and the test tube. The low copy number plasmids are exploited under certain conditions like the cloned gene produces the protein which is toxic to the cells. More stable because of circular configuration. Foreign DNA attached to ori also begins to replicate. leucinopine, nopaline, octopine, etc. Introduction of the ligated segment into the host cell for propagation. No upper limit on DNA that can be cloned. The DNA insert size varies between 150 to 350 kb. Plasmids only encode those proteins which are essential for their own replication. Usually, a high-copy vector is … The whole process of molecular cloning involves the following steps: A cloning vector is also a fragment of DNA which is capable of self-replication and stable maintenance inside the host organism. This DNA segment contains cohesive terminal sites (cos sites). For cloning vectors only: Copy Number . An inducible lac gene promoter is present. It should be capable of working under the prokaryotic as well as the eukaryotic system. They are way more efficient than plasmids for cloning large inserts. The vector needs to be a DNA molecule so that it can be cloned with the gene of interest. For easy incorporation into the host machinery, a vector should itself be small in size and be able to integrate large size of the insert. Retroviral vectors are used for introduction of novel or manipulated genes into the animal or human cells. Detection easy because of antibiotic-resistant genes. A selectable marker, possibly an antibiotic resistance gene, must be present to screen the recombinant cells. Examples: pBR322, pUC18, F plasmid, Col plasmid. Characteristics of Plasmid vector: They are widely used as a tool to study and analyze oncogenes and other human genes. It can be extracted from a virus, plasmid or cells of a higher organism. The plasmids have high copy number which is useful for production of greater yield of recombinant plasmid for subsequent experiments. Most of the cloning vectors are genetically engineered. They are present in bacteria, archaea, and eukaryotes. This foreign segment of DNA is replicated and expressed using the machinery of the host organism. Digestion of DNA fragments of the target segment and the vector DNA with the help of restriction enzymes, Ligation of the target segment with the vector DNA with the help of DNA ligases, and. It must be self-replicating inside host cell, It must possess restriction site for Restriction Endonuclease enzymes, Introduction of donor DNA fragment must not interfere with replication property of the vector, It must possess some marker gene such that it can be used for later identification of recombinant cell, Marker gene: Ampicillin and Tetracycline resistant gene, Restriction site for various restriction endonucleases, It is modification of bacterial F-plasmid, Marker gene: chloramphenicol resistant gene and lactose metabolizing gene (LacZ), It is an artificial chromosome having yeast centromere isolated from, Marker: similar as for identification of yeast cell, It can recombinant only 4-5 Kbp of donor DNA fragment, Example: Eukaryotic expression vector pSG5, An eukaryotic vector modified such a way that it can be expressed in prokaryotic cell known as expression vector, it allows RNA polymerase to transenecribe g, It has combined feature of both phase and plasmid, It is artificially synthesized chromosome used to transfer human gene, Cloning limit; No limit, it can carry large segment of DNA,,,,

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