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Transcript
CLONING VECTORS Shumaila Azam CLONING VECTORS The DNA into which a foreign piece of DNA is cloned is called a ‘VECTOR’ • Vectors are those DNA molecules that carry a foreign DNA fragment when inserted into it. • Vectors are also known as vehicle DNAs. • Based on the nature and sources the vectors are grouped into bacterial bacteriophages, plasmids, cosmids and phasmids. TYPES OF VECTORS If a vector is used for reproducing the DNA fragment, it is called a "cloning vector". If a vector is used for expressing certain gene in the DNA fragment, it is called an "expression vector". CLASSES OF VECTORS • Plasmids: Accept up to ~10 kb ‘foreign’ DNA • Phage : 5-20 kb fragments (its own genome is only 50 kb!) Commonly used in making genomic libraries. (very high efficiency of transfection) • Cosmids: 35-45 kb – similar to plasmids (high efficiency for transformations). Cosmids are hybrids of phages and plasmids. • YACs (Yeast Artificial Chromosomes): 300-2000 kb! (essential for cloning very large fragments) PLASMIDS PLASMIDS • Plasmids are self-replicating, double stranded, circular DNA molecules that are maintained in bacteria as independent extra chromosomal entities. • Virtually all bacterial genera have plasmids. • The size of plasmids ranges from a few kb to near 100 kb. STRUCTURE OF PLASMID It contains a polylinker which can recognize several different restriction enzymes, An antibiotic-resistance gene for selective amplification A replication origin (ORI) for proliferation in the host cell. Structure of a typical plasmid CLASSES OF PLASMIDS • Plasmids have basic attributes to make them potential vectors for carrying cloned DNA. • Some plasmids carry information for their own transfer from one cell to another called F plasmids. • Some plasmids encode resistance to antibiotics and are called as R plasmids. CLASSES OF PLASMIDS • Some plasmids carry specific sets of genes for the utilization of unusual metabolites and are called as degradative plasmids. • Some plasmids have no apparent functional encoding genes and are called as cryptic plasmids. • Plasmids can range in size from less than 1 to more than 500kb. • Each plasmid has a sequence that functions as an origin of DNA replication, with out this site, it cannot replicate in a host cell. CLASSES OF PLASMIDS • Some plasmids are represented by 10 to 100 copies per host ,these are called high copy number plasmids. • Some plasmids maintain 1 to 4 copies per cell and are called low copy number plasmids. • Seldom does the population of plasmids in a bacterium make up more than approx 0.1 to 5.0 percent of the total DNA. • When two or more types of plasmids cannot coexist in the same host cell, they are said to belong to a single incompatibility group. CLASSES OF PLASMIDS • But plasmids from different incompatibility groups can be maintained together in the same cell. This coexistence is independent of the copy numbers of the individual plasmids • Some plasmids, because of their specificity of their origin of replication , can replicate in only one species of the host cell and are called narrow range plasmids. • Other plasmids have less specific origins of replication and can replicate in a number of bacterial species and are called broad range plasmids. IMPORTANT FEATURES OF PLASMIDS • A small size, which is necessary because the efficiency of transfer of exogenous (foreign) DNA into E.coli decreases significantly with plasmids that are more than 15kb long. • Unique (single) restriction endonuclease recognition sites into which the insert DNA can be cloned • One or more selectable genetic markers for identifying recipient cells that carry the cloning vector --- insert DNA construct. IMPORTANT FEATURES OF PLASMIDS • Consequently the plasmid cloning vectors have to be generally engineered. • However naturally occurring plasmids often lack several important features that are required for high quality cloning vector. THANKYOU