Genetic engineering techniques of such gene manipulation involve
- Isolation of a specific deoxyribonucleic acid (DNA) molecule Or molecules to be replicated (the passenger DNA)
- The joining of this DNA with a DNA vector (also known as a vehicle or a replicon). It is capable of autonomous replication in a living cell after foreign DNA has been inserted into it
- The transfer, via transformation or transfection. of the recombinant molecule into a suitable host.
- Isolation of passenger DNA
Passenger DNA may be isolated in a number of ways;
(a) DNA restriction: The most common method of isolation of passenger DNA is DNA restriction. Restriction endonucleases break the high-molecular-weight DNA into fragments. Three different classes of the 4.! enzymes are present. But only type II restriction endonuchases are used extensively in the manipulation of DNA Type II restriction endonucleases are DNAases. They recognize specific short nucleotide sequences
(usually 4 to 6 base pairs in length), and then cleave both strands of the DNA duplex. It generates discrete DNA fragments of defined length and sequence.
The various fragments of a specific DNA are separated into different bands of distinct molecular weights by agarose gel electrophoresis. Specific sequences of these bands can be identified by a technique called Southern blotting. In this technique. DNA restriction fragments present on a gel are denatured. They are blotted on a nitrocellulose filter. The filter is incubated together with a radioactively labeled DNA or RNA probe. These probes are specific for the gene under study. The labeled probe hybridizes to its complement in the restricted DNA. The regions of hybridization are detected by autoradiography. Fragments of interest can then be separated out of these gels and used for cloning.
(b)Sonification: It is another method used to generate small DNA fragments by mechanical shearing. In this case, intense sonification of high-molecular-weight DNA is done with the help of ultrasound or high-speed stirring blender. This method is used to produce DNA fragments of a certain size range. Shearing results in random breakage of DNA. It produces termini consisting of short, single-stranded regions.
(c)Complementary DNA: Other source of DNA is synthesis of complementary DNA (cDNA). It is synthesized in test tube artificially by joining nucleotides.
- Joining DNA molecules
following techniques are used to join DNA molecules
(a) The cleavage with a restriction endonuclease creates cohesive ends (sticky ends). These fragments can be jointed with a similarly cleaved DNA from another source like vector. The joint has nicks a few base pairs apart in opposite strands. The enzyme DNA ligase is used to repair these nicks. It forms an intact duplex recombinant molecule. Cohesive ends can also be created by the addition of synthetic DNA linkers to blunt-ended DNA molecules.
Transformation in E-Coli
The desired DNA sequence attached to a DNA vector is transferred to a suitable host. Transformation is defined as the introduction of foreign DNA into a recipient cell. Transformation of a cell with DNA from a virus is called transfection.
Transformation in any organism involves ( I ) a method that allows the introduction of DNA into the cell and (2) the stable integration of DNA into a chromosome, or maintenance of the DNA as a self-replicating entity.
Transformation in microorganisms
(a)Escherichia coil is the host of choice for cloning experiments.
The transformation of E. con is an essential step in these experiments. Escherichia call are treated with calcium chloride. They then take up DNA from bacteriophage lambda and other plasmid DNA. Calcium chloride causes some structural alterations in the bacterial cell wall.
(b) Transformation in Bacillus species is done by polyethylene glycol-induced DNA uptake in bacterial protoplasts. The bacterial cell wall is later regenerated. Actinomycetes can be similarly transformed.
(c) Transformation can also be achieved by first entrapping the DNA with liposomes. Later these liposomes are fused with host cell membrane. Similar transformation methods have been developed for lower eukaryotes such as the yeast Saccharomvces cerevisiae and the filamentous fungus Neurospora crassa.
Transformation in animals
(a) Several methods are available for the transfer of DNA into cells of higher eukaryotes. Specific genes or entire viral genomes can be introduced into cultured mammalian cells in the form of a coprecipitate with calcium phosphate. DNA complexed with calcium phosphate is readily taken up and expressed by mammalian cells.
(b) DNA complexed with diethylamino-ethyl-dextran (DEAFdextran) or DNA trapped in liposomes or erythrocyte ghosts may also be used in mammalian transformation.
(c) Bacterial protoplasts containing plasmids can be fused to intact animal cells with the help of chemical agents like polyethylene glycol (PEG).
(d)DNA can be directly introduced into cells by microinjection. The efficiency of transfer by each of these methods is quite variable.. Transformation in plants
(a)In plants, (T)-DNA region of the tumor-inducing (Ti) plasmid of Agrobacterium tumefaciens are used to introduced DNA into plant cells.
(b) But there are limitations of the host range of A. tumefaciens. Therefore, alternative transformation systems are developed for
gene transfer in plants. They include the use of liposomes and induction of DNA uptake in plant protoplasts.
(c) Foreign DNA is also introduced into plant cells by a technique called electroporation. This technique involves the use of electric pulses to make plant plasma membranes permeable to plasmid DNA molecules. Plasmid DNA taken up in this way has been shown to be stably inherited and expressed.
(d) Use of particle gun: This method is developed by John C Sanford and Theodore M. Klein of Cornell University in 1987. They introduced DNA into a plant tissue culture callus. They developed a device called particle gun. This gun bombards a callus with microscopic metal particles. These particles are coated with DNA. The callus produces genetically altered somatic embryos. These embryos produce adult plants. Many plants like corn and wheat varieties are genetically engineered by this method
- Cloning vectors
There is a large variety of potential vectors for cloned genes. The vectors differ in different classes of organisms.
(a) Prokaryotes and lower eukaryotes: Three types of vectors are used in these organisms: plasmids, bacteriophages, and cosmids.
- Plasmids: Plasmids are extrachromosomal DNA sequences that are stably inherited. Escherichia coli and its plasmids constitute the most versatile type of host-vector system known for DNA cloning. Several natural plasmids, such as ColEI, have been used as cloning vehicles in E. coll. In addition, a variety of derivatives of natural plasmids have been constructed by combining DNA segments and desirable qualities of older cloning vehicles. The most versatile and widely used of these plasmids is pBR322. Transformation in yeast has been demonstrated using a number of plasmids, including vectors derived from the naturally occurring plasmid of yeast.
- Bacteriophage: Bacteriophage lambda is a virus of E. coll. Several lambda-derived vectors have been developed for cloning in E coll. These lambda derivatives have several advantages over plasmids: (I) Thousands of recombinant phage can easily be screened for a particular DNA sequence on a single petri dish by molecular hybridization. (2) Packaging of recombinant DNA in laboratory cultures provides a very efficient means of DNA uptake by the bacteria. (3) Thousands of independently packaged recombinant phages can be easily replicated and stored in a single solution as a “library– of genomic sequences.
- Cosmids: Plasmids have also been constructed that contain the phage cos DNA site and ColE1 DNA segments. Cos DNA is required for packaging into the phage particles. ColE I DNA segments required for plasmid replication These plasmids are called as cosmids. The recombinant cosmid DNA is injected into a host. They circulate like phage DNA but replicates like plasmid.
(b)Animal cells: Very few vectors are available for introducing
foreign genes into animal cells. The most commonly used are derived from simian virus 40 (SV40). Normal SV40 cannot be used as a vector. However. 5V40 mutants lacking portions of the genome can be used as vector.
(c)Plant cells: Two systems for the delivery and integration of foreign genes into the plant genome are the Ti plasmid of the soil bacterium Agrobaclerium and the DNA plant virion cauliflower mosaic virus. ‘Me Ti plasmid is a natural gene transfer vector carried by A. tumefaciens, a pathogenic bacterium. It causes crown gall tumor formation in dicotyledonous plants. A 1-DNA segment present in the Ti plasmid becomes stably integrated into the plant cell genome during infection.