Different Types of Vectors & Their Advantages & Disadvantages In rDNA Technology

Vector is the central component of a gene cloning process. It is a small piece of DNA into which
a foreign DNA fragment can be inserted. The insertion of the fragment is carried out by treating
the vector and the foreign DNA with a restriction enzyme that creates the same overhang, then
ligating the fragments together.

Types of Cloning Vectors:

 Plasmid Vectors
 Bacteriophage Vectors
 Cosmids
 Phagemids
 Bacterial Artificial Chromosomes(BAC) & Yeast Artificial Chromosomes
(YAC)

Plasmid Vectors: Plasmid vectors are double-stranded, extra-chromosomal DNA molecules,

which is circular and self-replicating.

Features:

 Plasmid is a double-stranded circular and supercoiled DNA.

 Within a cell, it can exist autonomously. It can replicate independently of
the bacterial chromosome.
 It has a molecular weight of 106-108 which may encode from 40-50 genes.
 It has about 1-3% of the weight of the bacterial chromosome consisting of
1500-400,000 base pairs.
 Plasmid as large as 2 million base pairs can occur in some bacteria.
Types of plasmids: Based on their function, plasmids are of five types:

 Resistance plasmid

 Fertility plasmid

 Bacteriocinogen or Col plasmid

 Degradative plasmid

 Virulence plasmid

Advantages:

 It is small and easy to handle

 Easy purification can be made
 Straightforward selection strategies
 Useful for cloning small DNA fragments (< 10kbp)
 Readily isolated from cell

Disadvantages:

 Less useful for cloning large DNA fragments (> 10kbp)

Bacteriophage Vectors: Phage vectors consist of an essentially complete phage genome, often
M13 phage, into which is inserted DNA encoding the protein or peptide of interest. Typically,
the remainder of the phage genome is left unchanged and provides the other gene products
needed for the phage life cycle.
Phages are two types:

 Lambda Phage
 M13 Phage

Advantages of Lambda Phage:

 Large DNA fragments upto 23kb can be carried

 Efficiency of gene transfer is high

Disadvantages of Lambda Phage:

 rDNA may enter lytic cycle

 Narrow host range

Advantages of M13 Phage:

 Blue/white screening system

 Genes cloned in pUC18 or pUC19
 Can be subcloned to same sites in M13mp equivalent
 Different directions for multiple cloning sites
 Both strands of cloned DNA converted to single stranded form in different
vectors

Disadvantage of M13 Phage:

  Limits to size of cloned DNA (2 kb)
 Low yield of DNA
 Cannot amplify phage genome numbers much
 Phage proteins toxic in high concentrations

Cosmid Vectors:

Cosmid is a hybrid DNA formed by the joining of a plasmid and lambda phage DNA carrying
aCos site (cohesive). In brief cosmid is a plasmid carrying the Cos Site of a L-phage DNA.
Cosmidis not a naturally

found in living cells. It is constructed vector. E.g. Col E1 cosmid is a typicalcosmid used in
genetic engineering.

Advantages of Cosmids:

 Large DNA fragments

 Used to establish gene libraries of lower and
higher organisms
 Gene cloning through cosmid helps in the
study of non-sense seq in the genome of
organism

Disadvantage of Cosmids:

 The packaging enzyme fails to pack rec cosmid into phage heads if 1 of cos-sites is
missing

Phagemids Vectors:

A phagemid or phasmid is a DNA-based cloning vector, which has both bacteriophage and
plasmid properties. These vectors carry, in addition to the origin of plasmid replication, an origin
of replication derived from bacteriophage.
 Advantages of Phagemid:

 PHAGEMIDS can be maintained as plasmids or phage particles in E.coli

 The desired gene can be integrated into chromosomal DNA of E.coli using phagemids
 Plasmid portion can be released free from a phagemid after rec.phagemid is introduced
into an E.coli Strain
 The phages having rec.phagemids can be stored easily for a long time.

Artificial chromosome vectors:

Artificial chromosomes are DNA molecules assembled in vitro from defined constituents, which
guarantee stable maintenance of large DNA fragments with the properties of natural
chromosomes.

Artificial chromosomes are useful for genome sequencing programmes, for functional
characterization of entire genomic regions and for the transduction of large DNA segments into
human and nonhuman mammalian cells.

 Bacterial artificial Chromosome (BAC): Engineered DNA molecule used to clone DNA
segments in bacterial cells up to 300kb.
 Yeast Artificial Chromosome (YAC): Engineered chromosome derived from the DNA of
the yeast, cloning very large (1000-2000kb) DNA segments.

Advantages:
  Useful for cloning extremely large DNA fragments
 Very important for genome sequencing projects

Disadvantages:

 Not easy to handle

 YAC is very fragile and prone to breakage
 YAC is Unstable, with their foreign DNA inserts often being deleted
 Loss of the entire YAC during mitotic growth
 Difficult to separate the YAC from the other host chromosomes
 The yield of DNA is not high
 Chimaerism