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Gene Cloning Vectors: Different Types of Vectors, Essential Features of an Ideal Vector and pBR322
Gene cloning vector are small plasmid, phage or animal virus DNA molecules used to transfer a DNA fragment from a test tube into a living cell. Cloning vectors are used for obtaining millions of copies of cloned DNA fragment. The cloned genes in these vectors are not expected to express themselves at transcription or translation level.
Cloning vectors (Cloning vehicles) are used for creating genomic library or preparing probes or genetic engineering or other basic studies.
The most important requirement for recombinant DNA technology is the cloning and expression vectors The recombinant DNA is produced by cloning a foreign DNA isolated either from the genome or synthesized chemically or as cDNA using mRNA molecule. The cloning of this DNA can be done only when another DNA molecule is available that may replicate in the transformed host cell. The other DNA molecule used for joining the foreign DNA is celled Vectors.
Vecors may be plasmids, cosmids, phagemids, transposons, a virus, YAC or BAC.
Following are the desirable features for a cloning vector:
- Small size.
- Should contain origin of replication.
- Should have unique restriction sites as polylinkers or multiple cloning site (mcs).
- Should have one or more marker genes for identification ad isolation of subpopulation of bacteria containing vector.
- Should have relaxed mode of replication.
- The vector must be able to replicate in host cells.
Essential Features of an Ideal Vector
Different Types of Vectors
Insert Size (kb)
9 22 kb
33 47 kb
75 350 kb
100 1000 kb
- The plasmid pBR 322 is derived vector to transfer derived genes to E.coli.
- It was constructed by Boliver and Rodriguez.
- pBR: P stands for plasmid; B and R refer to the first letters of the scientists.
- It is 4.3 kbp in size.
- It has an origin of replication, tetracycline resistance gene(Tetr) and amphicillin resistance gene (Amp r).
- Tetr gene has unique sites for six restriction enzymes
- The Amp r gene has unique sites for three restriction enzymes( ScaI, PuvI, and PstI)
- The origin of replicationn , which is derivative of ColE1 plasmid, helps for the replication of the plasmid in E.coli.
- The desired gene is inserted at the Tetr or Ampr hene of the plasmid.
- The recombinant pBR 322 is delvered into E.coli by means of bacterial transformation.
- Transformed E.coli cells are selected by means of insertional inactivation of the antbiotics resistant gene.
- The recombinant PBR322 plasmid is useful for cloning 5-10 kbp long DNA in E.coli
pBR322 Gene Cloning Vector Explained
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