What is genetic engineering?
In a nutshell, transfer of a desirable gene or genes from an organism with or without modifications to a selected target organism to transfer some specific characteristics to the target or to replace its defective genes with help from enzymatic tools is genetic engineering or gene manipulation.
If such transfer is done in human to rectify some genetic defect, it is called gene therapy. The technique involves production of DNA lengths which have some foreign segment tagged to them. Such DNA lengths would be called recombinant DNA (r-DNA). That is why genetic engineering is also referred as r-DNA technology. The figure given below presents a gene transfer schedule from a human cell to a bacterial cell producing an r-plasmid.
Gene cloning
To obtain several copies of a gene, one method involves transfer of that gene into a bacterial cell by joining it to a plasmid and providing suitable culture conditions to the bacterial cell to propagate by asexual binary fission to form large colonies of bacteria. Each bacterial cell of the colonies of thousands of cells would have a copy of that gene. This technique is called gene cloning. Stock of genes cloned as such is called gene library. First gene was cloned in 1973 by Herbert Boyer and Stanley Cohen in Stanford university, California by this technique.
Click here to see a diagram showing the basic method of gene transfer
Tools and techniques of genetic engineering
Tools used in genetic engineering are a group of enzymes and reagents that are used to isolate, purify, cut, synthesize, modify, join together and transfer required DNA segments among organisms. Another group of important tools is that of vectors required as intermediate carriers to transfer a gene from one source to another. Chief enzymes that are required for the purpose are:
• Restriction endonucleases
• DNA ligase
• Exonucleases
• DNA polymerases
• Alkaline phosphatase
• Polynucleotide kinase
• Terminal deoxynucletidyl transferase
• Reverse transcriptase
Restriction endonucleases or restriction enzymes are special types of endonucleases that cut DNA at specific sites or motifs. They are able to recognize their site in DNA and act there. Restriction enzymes are obtained from bacteria which use them to prevent foreign phage DNA from parasitizing by digesting it on its specific sites. In genetic engineering experiments, type II enzymes of this group used as they are very specific and cut DNA within recognized motifs which are mostly 4-7 bases long. The motifs have two-fold symmetry called palindromes. For example, the enzyme Eco RI is able to identify 5’GAATTC3’ motif of DNA molecules and cut between G and A. As such, a DS DNA would have staggered cuts i.e. at two different points on the two strands.
Likewise, Bam HI acts on the motif 5’GGATCC3’ causes cut in both DNA strands between GG.
Click here to see the diagram
Some restriction enzymes cut both strands of DNA at the same point e.g. Hin dII which acts at the motif 5’GTCGAC3’ and cuts the strands between and CG.
Click to see the diagram
The naming of a restriction enzyme is based on the scientific name of the bacterium from which it has been obtained, its strain and the serial number of the enzyme from that source. Eco RI was obtained from E. coli strain R before any other enzyme was obtained from this bacterial strain (hence, serial number I). Some other common restriction enzymes are Sal I, Eco RV, Hae I etc. with their specific restriction sites. DNA ligase is the enzyme used for joining two DNA lengths. This enzyme forms 5’-3’ phosphodiester bond from the 5’- phosphate end of a chain to the 3’- OH end of another chain. Exonucleases differ from endonucleases in digesting nucleotides of a DNA chain one by one either from its 5’ or from 3’ end. These enzymes are used to suitably modify DNA chain ends. Some exonucleases have multiple activities. For example, exonuclease III obtained from phage-infected E. coli cells removes nucleotides of a DNA strand one by one starting from its 3’ end. It can also be used to add a phosphate group to the 3’ end of an ss DNA or ds DNA. It is also able to digest DNA-RNA hybrid molecules. DNA polymerases are used to synthesize small or large DNA lengths as per requirement. DNA polymerase I and T4 DNA polymerase are commonly used polymerases. Klenow fragment is the name of the DNA polymerase I from E. coli without exonuclease activity which this enzyme utilizes to replace RNA primers during DNA replication in bacterial cells. Alkaline phosphatase is used to remove phosphates from the 5’ end of an ss DNA length or from an RNA length. Polynucleotide kinase is the enzyme that can transfer a phosphate group from an ATP molecule to a DNA or RNA. It is obtained from phage-infected E. coli. Terminal transferase is a kind of DNA polymerase which is capable to add given nucleotides to a 3’-OH end of a DNA length. It does not need a template for complementary copying as generally required by DNA polymerases. Reverse transcriptase which is also a kind of DNA polymerase but RNA dependent is used when c-DNA (complementary DNA) is to be produced from a given m-RNA length. Vectors are vehicles by whose help genes are transferred between organisms. Their suitability depends on the type of organism to which a gene is to be transferred. Several vectors have been designed by genetic engineers for bacteria, animal species and plants. For gene transfer into bacterial cells, suitable vectors are plasmids and bacteriophages. Bacteriophages are used due to their natural ability to transfer their genetic material to bacteria. If you tag your desirable foreign gene to the DNA of the phage, it would easily be delivered into its host bacterial cell. Several advanced and more efficient plasmid vectors have been artificially developed by modifying original bacterial plasmids through genetic engineering tools themselves. Some such plasmid vectors are pBR322, pCR1, pUC, pMB9 etc. Cosmids are genetically modified plasmids with lambda phage gene sequence which make them able to package into lambda phage head. Foreign genes tagged to a cosmid and packaged into a ? phage can be delivered into E. coli cells. Suitable vector for gene transfer into plants is Ti plasmids or their modified segments which have ability to enter and harbour plant cells. Ti plasmids are tumour inducing plasmids found in the soil bacteria which are species of the genus Agrobacterium. One common species is Agrobacterium tumefaciens which is able to induce crown galls (tumour like bodies) in dicot plants after transferring its Ti plasmid to the host cell. Tumour is caused due to the activity of genes in T DNA (tumour DNA) segment of the plasmid. Modification and recombinant types of simian virus 40, adenoviruses, retroviruses and other animal viruses are used as vector for transfer of genes into animal cells.
Joining two DNA lengths together
Two ds DNA lengths can be joined together if they have sticky tails (extensions having complementary bases respectively). If you cut out gene lengths from two sources to join together, you may use a common restriction enzyme for both causing staggered cut like that by Eco RI. Both lengths would have complementary sticky tails that can base pair if provided with suitable conditions in vitro. Fig. 4 shows the two fragments of DNA which Eco RI would create respectively from both the resources.
Click here to see the diagram
In case of blunt ends as created by Hin dII, sticky tails are created artificially by adding complementary nucleotides at 3’ ends of one strand each of the two DNA lengths to be joined with the help of the enzyme terminal transferase. Sometimes, if need be, blunt ends are created by the use of exonucleases.
Polymerase Chain Reaction (PCR)
Polymerase Chain Reaction (PCR) technique was developed by Kary Mullis in 1985 at the Cetus Corporation in Emeryville, California. This technique is able to amplify a selected segment of DNA into thousands of copies in a very short time. It is a better alternative to gene cloning for this purpose. It involves a programmed machine called thermal cycler which can alternate high and low temperatures in a cyclic manner in seconds. Desired segment of DNA is amplified through recurrent replication process. As in case of natural DNA replication, forward and reverse primers are required for replication. Primers used here are ss DNA strands complementary to the 3’-5’ extremes of the ds DNA length which is to be copied. Target DNA is put up with primers of 20-30 base pairs, a suitable buffer solution, free DNA nucleotides (d ATP, d GTP, d CTP and d TTP) and reagents MgCl2/KCl and a DNA polymerase enzyme in a reaction tube kept in the PCR machine. DNA polymerase used is TAQ polymerase obtained from the thermal bacterium Thermus aquaticus or other suitable polymerase which does not degrade at high temperatures such as 950C. In just 25 cycles of PCR, more than a million copies of the desired DNA segment are produced.
Look to Fig. 5 to grasp how PCR functions.
Conclusion
When you introduce foreign genes into the genome of an organism, it gets genetically modified. Genetic modification would be reflected in its phenotype or functions. Several trangenic organisms have been created. Some transgenic plant varieties are those of tobacco, potato, cauliflower, grapes, cotton, papaya, apple, rice, pear, rye, corn etc. Transgenic tomato is able to withstand the herbicides due to bxn gene from the bacterium Klebsiella. Bt maize, Bt soya and Bt cotton varieties have some genes transferred from the bacterium Bacillus thuringiensis. These genes produce proteins that kill some phytophagus insects resulting in greater yields of the crops. A genetically engineered strain of Pseudomonas putida bacterium (called superbug) has ability to degrade petroleum components and is used to clear oil spills by on-sea tankers carrying petroleum. Dr. Anand Mohan Chakrabarty (1979) developed the superbug by accumulating together genes capable to produce enzymes that can degrade octane, hexane, decane, xylenes, toluenes etc. in one recombinant cell of P. putida. There are now several successful examples of genetically engineered useful organisms. Transgenic animals have been obtained in mouse, chicken, cow, fish, goat, sheep etc. These animals can utilize their feed more efficiently, produce products like milk or meat with better percentage of useful components or of better quality. Food items prepared from genetically modified resources are called GM foods. The list of achievements in the area of genetic engineering is quite long and is growing day by day. Gene replacement therapy (G.R.T.) is an area directly related to human health. The technique can replace defective genes of human by correct forms of genes from other sources or synthesized artificially.