Transcript lect 5- Cloning Vectors

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.
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