GENE CLONING PLASMIDS COSMIDS cloning vectors, cosmid properties, gene cloning, plasmid properties, properties, quality of good vectors, vectors pbr322 , pbr325, puc18, puc19, puc series cosmid col

1. Gene Cloning
Plasmid Properties and
Cosmid Properties
BY:
NOOR ARSHIA
4TH SEM Bsc(MB)

2. Concept Of Gene
Unit of function
• Recombination
• Mutation
Gene is considered to control
the inheritance of one
character

3. Gene Cloning
Cloning: The generation of a large number of
genetically identical molecules is
called cloning.
Gene Cloning is a molecular biology technique
that makes many identical copies of a piece of
DNA such as gene.
Gene cloning is a process in which a gene of
interest is located and copied out of a DNA
extracted from an organism.

4. TOOLS USED FOR GENE
CLONING
1. NUCLEASES
Example : Restriction endonuclease
2. LIGASES
Example: DNA ligase
3. POLYMERASES
Example : Reverse transcriptase
4. DNA Modifying enzymes
Example : Alkaline Phosphate

5. CLONING VECTORS
A vector is a DNA molecule which has an ability to replicate in a
appropriate host cell into which the DNA fragment is to be
cloned.
A vector must have an origin of replication ( ori ) that functions in
the host cell
TYPESOF CLONINGVECTORS
Any extra-chromosomal small genome can act like a cloning
vector.
 PLASMID
 BACTERIOPHAGE
 VIRUS
 COSMID
 ARTIFICIAL CHROMOSOMES

6. • Production and Isolation of DNA fragments to be
cloned.
• Insertion of the isolated gene into a suitable vector.
• Introduction of recombinant DNA into the host.
• Selection of the transformed host cell.
• Identification of the clone containing desired gene.
• Multiplication of introduced gene in the host.
• Transfer and expression of gene into another
organism.
Steps in Gene Cloning

14. PROPERTIES OF A GOOD VECTOR
 Replicate Autonomously
 Small in size ( Less than 10kb )
 Easy to isolate and purify
 Easy to introduce into host cells
 Must possess restriction endonuclease enzyme
 DNA donor fragment introduced must not interfere with replication
property of the vector.
 It must possess marker genes which can be used for later
identification of recombinant cell
 It must possess multi cloning sites

15. PLASMIDS
• A plasmid is a small, circular, extra-
chromosomal double stranded DNA that has
the capacity to replicate independently.
• Discovered by Laderberg in 1952.
• It naturally occur in bacteria, however
sometimes present in archaea and
eukaryotes.

16. PROPERTIES
• Specific to one or a few particular bacteria.
• Replicate independently and code for their own
transfer.
• Do not cause damage to cells and generally are
beneficial,
• Do not have extracellular forms and exist inside
cells simply as free and typically circular DNA.
• Size ranges from 1 kbp to several mbp.
• Number of plasmids in an individual cell may vary,
ranging from one to several hundreds, denoted by
copy number.

17. • Genes carried by plasmid encodes traits for
antibiotic resistance or resistance to heavy
metal.
• Some produces virulence factor that help in
defence or nutrient utilization.
• Plasmids can also provide bacteria with the
ability to fix nitrogen.
• Some also exhibits properties like sulphur
utilization, hydrocarbon degradation, drug
resistance etc.
PROPERTIES CONTINUED…

18. ELEMENTS OF PLASMIDS
• Origin of replication:
It is the DNA sequence
which directs initiation of plasmid replication by
recruiting bacterial transcriptional machinery.
• Antibiotic resistance gene:
These genes allows for
selection of plasmid containing bacteria by
providing a survival advantage to the bacterial
host.
• Multiplecloning site:
This is the short segment
containing several restriction enzyme sites,
enabling easy insertion of foreign DNA.

19. EXAMPLES
• pBR322
• pBR327
• pBR325
• pBR328
• pUC8
• pUC9
• pUC12
• pUC13

20. pBR322
• Is a hybrid plasmid
• It is an artificial plasmid formed
by combining 3 naturally
occuring plasmids
• P stands for plasmid
• BR represents the first letters
of scientist who constructed
this plasmid.
• 322 refers to the serial number
of the plasmid constructed in
that lab.

21. COSMID
A cosmid is a type of hybrid plasmid that contains
a Lambda phage cos sequence .
Cosmids (cos sites + plasmid = cosmids) DNA sequences
are originally from the lambda phage
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

23. THANK
YOU