Separation and isolation of DNA fragments in Recombinant DNA Technology

formation-pores-agarose-gel

Gel electrophoresis
• After the cutting of DNA by restriction enzymes, fragments of DNA are formed. These fragments can be separated by a technique called gel electrophoresis.
• Electrophoresis is a technique of separation of charged molecules, under the influence of an electrical field, so that they migrate in the direction of electrode bearing the opposite charge, through a medium/matrix.
• The most commonly used matrix is agarose, which is a polysaccharide extracted from sea weeds.
• DNA fragments separate according to size, through the pores of agarose gel.
• The separated DNA fragments can be seen only after staining DNA with a compound ethidium bromide (EtBr), as bright orange coloured bands, on exposure to UV radiations.
• The separated bands of DNA are cut out from the agarose gel and extracted from the gel piece. This step is called as elution.

typical agarose gel electrophoresis

Cloning Vectors (Vehicle DNA)
• The vectors are DNA molecules that can carry a foreign DNA segment and replicate inside the host cell.
• Vectors may be plasmids, bacteriophages, cosmids. Yeast artificial chromosomes (YACs), Bacterial artificial chromosomes (BACs), transposons, and virus.
• Out of these vectors, plasmid vectors and bacteriophage vectors are commonly used.
Plasmid vectors
• Plasmid vectors are extra-chromosomal, self- replicating, usually circular, double-stranded DNA molecules, found naturally in many bacteria and also in some yeast.
• They were discovered by William Hays and Joshua Lederberg in 1952.
• Plasmids are usually not essential for normal cell growth and division, but they often confer some traits on the host organism e.g., resistance to certain antibiotics.
• The plasmid molecules may be present as 1 or 2 copies or in multiple copies (500-700) inside the host organism.
• The naturally occurring plasmids have been modified to serve as vectors in the laboratory.
• The most widely used, versatile, easily manipulated vector pBR322 is an ideal plasmid vector.

pBR322 vector

Diagram showing essential features of Plasmid pBR322

• This was the first artificial cloning vector developed in 1977 by Boliver and Rodriguez.
Nomenclature
In pBR322 plasmid:
• p – denotes that it is a plasmid.
• BR – stands for Boliver and Rodriguez, who developed this plasmid.
• 322 – is a number given to distinguish this plasmid from others developed in the same laboratory, e.g., pBR325, pBR327, pBR328, etc are other plasmids.
Structure
pBR322 plasmid contains:
• Antibiotic resistance genes such as ampicillin resistance (amp”) gene and tetracycline resistance (tetR) gene.
• Unique Cloning sites (Recognition sites) for restriction endonucleases.
• Plasmid pBR322 also possesses a variety of unique recognition sites for restriction endonucleases.
• Two unique sites, Pstl and Pvul are located with the amp” gene and BamHI, Sa/I, etc. are within tetR gene.
• Some other unique restriction sites are EcoRI, C/al, H/ndlll, Pvull. rop codes for the proteins involved in the replication of the plasmid.
Bacteriophage vectors
• Bacteriophages are viruses that attack bacterial cells by injecting their DNA into these cells.
• The injected DNA is selectively replicated and expressed in the host bacterial cell resulting in a number of phages, which burst out of the cell (lytic pathway) and reinfect neighbouring cells.
• Several bacteriophages that have been extensively modified for development of cloning vectors are lambda (X) phage and M13 phage.
• Lambda phage vector has a double-stranded, linear DNA genome of 48, 502bp, in which the 12 bases at each end are unpaired, but complementary.
• These ends are, therefore, sticky or cohesive and are referred to as the cos sites (cohesive end sites).
• These sites are important for packaging DNA into phage heads.
• An important feature of Lambda (X) genome is that a large fragment in the central region of its genome is not essential for lytic infection of E.coli cells.
• Therefore, vectors have been designed such that this region can be substituted or replaced by a foreign DNA.
• These vectors allow cloning of DNA fragments upto 23 Kb length.
• M13 phage vector is filamentous phage which infects E. coli having F-pili. Its genome is a single stranded, circular DNA of 6407 bp. Foreign DNA can be inserted into it, without disrupting any of the essential genes.
• After the M13 phage DNA enters the bacterial cell, it gets converted to a double-stranded molecule known as the replicative form RF, which replicates until there are 100 copies in the cell.
• The major advantages of developing vectors based on M13 are that its genome is less than 10Kb length, and can be purified and manipulated exactly like a plasmid.
Cosmid (cos-plasmid) vectors
• Cosmids have been constructed by combining certain features of plasmid and the ‘cos’ sites of phage Lambda (X).
• The simplest cosmid vector contains a plasmid origin of replication, a selectable marker, suitable restriction enzyme sites and the Lambda cos’ site.
• Cosmids can be used to clone DNA fragments upto 45 kbp in length.
• They can be packaged into X-particles. This is more efficient than plasmid transformation.
Bacterial artificial chromosome (BAC) vectors
• These vectors are based on natural, extra-chromosomal plasmid of £ coli.
• A BAC vector contains genes for replication and maintenance of the F-factor, a selectable marker
and cloning site.
• These vectors can accommodate upto 300-350 kbp (kilo base pairs) of foreign DNA and are also being used in genome sequencing projects.
Yeast artificial chromosome (YAC) vectors
• These are used to clone DNA fragments of more than 1 Mb-Megabase pairs (10-6 bp) in size, therefore, they have been exploited extensively in mapping the large genomes, e.g., in the Human Genome Project.
• These vectors contain the telomeric sequence, the centromere and the autonomously replicating sequence from yeast chromosomes.
• They also contain restriction enzyme sites and genes which act as selectable markers in yeast.

Phagemid vector
• Phagemid is a composite structure made of bacteriophage and plasmid. They are used for carrying larger DNA sequences.
Animal and plant viral vectors
Viruses that infect plant and animal cells have also been manipulated such that they can be used to introduce foreign DNA into plant and animal cells in culture. These are known as plant and animal viral vectors.
• A vector based on Simian Virus 40 (SV40) was used in the first cloning experiment involving mammalian cells in 1979.
• At present, retroviral vectors are the most commonly used vectors for cloning genes in mammalian cells.
• Plant cells do not contain any plasmids. But two plasmids pTi and pRi present in the bacteria provide a naturally occurring transformation system. These plasmids transfer a part of their DNA, called T-DNA, into the genomes of most dicot and some monocot plants.
Transposons as vectors
• DNA sequences which change their location in the genome, and hence are said to be mobile, are called transposable elements or jumping genes.
• They can be excised from one locus, and become inserted at separate locus. In this manner, they are used as vectors.
• Transposons were first observed by Clintock (1951) in maize plants.
• The activator (Ac) and dissociator (Ds) are
popular transposons of maize, also called Ac-Ds Elements. The transposons of Drosophila are known as P-elements.
Shuttle vector
• They can exist in both the eukaryotic cell and E.coli. Such vectors are known as shuttle vectors.
• These vectors contain two types of origin of replication and selectable marker genes, one
type that functions in the eukaryotic cell, and another that functions in E. coli.
• An example of a shuttle is the yeast episomal plasmid YEp.
• In case of plants, a naturally occurring plasmid of the bacterium Agrobacterium tumefaciens called Ti plasmid has been suitably modified to function as a vector.
• Most of the eukaryotic vectors are shuttle vectors.

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