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Cloning vectors

Presented by : Dr. Ujjwal Layek

Assistant Professor

Department of Botany

Rampurhat College

 What is a cloning vector?

 A vector is a DNA molecule that is used to carry a foreign DNA into the host cell. It has the ability to self replicate and integrate into the host cell.  Vectors can be a plasmid from the bacterium, a cell from the higher organism or DNA from a virus. The target DNA is inserted into the specific sites of the vector and ligated by DNA ligase. The vector is then transformed into the host cell for replication. Term Meaning plasmid circular DNA found in bacteria; can replicate independent of chromosomal DNA vector modified plasmid that can be used to clone and express foreign DNA ori origin of replication; site from which DNA replication starts selectable marker gene in vector that enables selection of recombinants cloning site restriction enzyme recognition site (often within selectable marker); site at which foreign DNA is ligated

pUC19 is one of a series of plasmid cloning vectors created by Joachim Messing and co- workers. It is a circular double stranded DNA and has 2686 base pairs. pUC19 is one of the most widely used vector molecules as the recombinants, or the cells into which foreign DNA has been introduced, can be easily distinguished from the non- recombinants based on color differences of colonies on growth media. pUC18 is similar to pUC19, but the MCS region is reversed. Notably, it has an N-terminal fragment of β-galactosidase ( lacZ ) gene of E. coli. The multiple cloning site (MCS) region is split into codons 6 - 7 of the lacZ gene, providing for many restriction endonucleases restriction sites. In addition to β-galactosidase, pUC 19 also encodes for an ampicillin resistance gene (ampR).

pBR 322 is a plasmid and was one of the first widely used E. coli cloning vectors. Created in 1977 in the laboratory of Herbert Boyer at the University of California, San Francisco, it was named after Francisco Bolivar Zapata, the postdoctoral researcher and Raymond L. Rodriguez. The p stands for "plasmid," and BR for "Bolivar" and "Rodriguez." pBR 322 is 4361 base pairs in length and has two antibiotic resistance genes – the gene bla encoding the ampicillin resistance (AmpR) protein, and the gene tetA encoding the tetracycline resistance (TetR) protein. It contains the origin of replication of pMB 1 , and the rop gene, which encodes a restrictor of plasmid copy number. The plasmid has unique restriction sites for more than forty restriction enzymes. Eleven of these forty sites lie within the TetR^ gene. There are two sites for restriction enzymes HindIII and ClaI within the promoter of the TetR^ gene. There are six key restriction sites inside the AmpR^ gene.

A bacterial artificial chromosome ( BAC ) is an engineered DNA molecule used to clone DNA sequences in bacterial cells (for example, E. coli). BACs are often used in connection with DNA sequencing. Segments of an organism's DNA, ranging from 100,000 to about 300,000 base pairs, can be inserted into BACs. Common gene components repE for plasmid replication and regulation of copy number. parA and parB for partitioning F plasmid DNA to daughter cells during division and ensures stable maintenance of the BAC. A selectable marker for antibiotic resistance; some BACs also have lacZ at the cloning site for blue/white selection. T7 & Sp phage promoters for transcription of inserted genes.

A cosmid is a type of hybrid plasmid that contains a Lambda phage cos sequence. They are often used as a cloning vector in genetic engineering. Cosmids can be used to build genomic libraries. They were first described by Collins and Hohn in 1978. Cosmids can contain 37 to 52 (normally 45 ) kb of DNA, limits based on the normal bacteriophage packaging size. They can replicate as plasmids if they have a suitable origin of replication (ori): for example SV 40 ori in mammalian cells, ColE 1 ori for double-stranded DNA replication, or f 1 ori for single-stranded DNA replication in prokaryotes. They frequently also contain a gene for selection such as antibiotic resistance, so that the transformed cells can be identified by plating on a medium containing the antibiotic.

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