Genetic engineering is the process in which the new DNA manually added to an organism. Important enzymes involved in genetic engineering are:-
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The enzymes act upon genome and digest the base pairs on 5′ and 3′ ends of a single-stranded DNA or at single-strand nicks or gaps in double-stranded DNA.
Endonuclease acts upon the genetic material and they can cut at any point in ds-DNA, except the ends, but their action only takes place only at one strand of the ds-DNA.
They can cut both strands of DNA when foreign nucleotides are introduced into the cell. They cleave DNA to generate a nick with a 5′ phosphoryl and 3′ hydroxyl terminus.
- Two major types, Type I and Type II, are the restriction endonucleases.
- Type I recognizes a specific sequence of DNA molecule but cuts elsewhere.
- Type II makes cuts only within the restriction sites producing 2 single strands break, one in each strand
- The complementary sequences are called Palindrome sequences or palindromes. When reading from 5’→3′ both strands have same sequences i.e., GAATTC/CTTAAG.
- Some important restriction endonucleases are Bam HI (Bacillus amyloliquesfaciens H). Eco RI (Escherchia coli RY13), Hpa I (Haemophilus parainfluenzae) etc.
- The extensive list of restriction enzymes and their sequences were provided by Roberta(1983).
- SI nuclease degrades the single-stranded DNA and the single strand of double-stranded DNA with cohesive ends.
Mutz and Davis (1972) demonstrated that cohesive terminus of the cleaved DNA molecule could be covalently tied with E.coli DNA ligase and were able to produce recombinant DNA molecule.
- The ligase from E.coli and that encoded by T4 phage are extensively used for covalently joining restriction fragments.
- The main source to obtain DNA ligase is T4 phage and so the enzyme is called as T4 DNA ligase.
- E.coli DNA ligase used nicotinamide adenine dinucleotide (NAD) as a cofactor, while T4 DNA ligase requires ATP for joining reaction.
Reverse transcriptase is used to synthesize the copy DNA or cDNA by using mRNA as a template. It is very useful in the construction of cDNA clone bank.
- Temin et. al in 1960 proposed that in some cancer-causing animal viruses. RNA is the genetic material. Transcription of gene takes place by DNA polymerase directed by viral RNA.
- S.Mizutani, A.M.Temin, and D.Baltimore (1970) discovered information can also pass back from RNA to DNA.
- Retroviruses (RNA only) contain RNA-dependent DNA polymerase which is also known as reverse transcriptase.
According to Kornberg (1974), the enzyme polymerizes DNA synthesis on DNA template and also catalyzes a 5’→3′ and 3’→5′ exonucleolytic degradation of DNA.
- Kornberg et al (1956) in E.coli worked out DNA polymerase I with molecular weight 1,09,000 daltons, having a single polypeptide chain of 1,000 amino acid residues.
The DNA of vector is circular and when it was linearized to join with the foreign DNA, then a problem arises as cohesive ends bind itself instead of the other DNA fragment.
- Alkaline phosphates prevent self-ligation of the vector and increase the frequency of production of recombinant DNA molecules.
Important role of recombinant DNA technology
- Recombinant DNA technology is useful in knowing defective genes in the fetuses. some of the genes can also be repaired.
- The technology can solve disputes of parentage with more accuracy than blood grouping and other types of tests.
- Through genetic engineering, microbes are being converted into synthetic factories for the synthesis of important biochemicals like insulin, growth hormone, interferons, and others.
- Agrobacterium tumefaciens can be transformed with any gene and transferred to dicot plants so that any deficient biochemical pathway can be made part of crop yield like pulses. The technique cannot be used for cereals.
- There are several major crop plants all over the world. Of these, three major kinds of cereal and most of the oil and protein crops(e.g., Pulses, cotton, soybean) show inbreeding. Maize exemplifies an outbreeder. Other notable outbreeders include sunflower, some forage grasses and legumes, potato, sugarcane and some orchard fruit crops, The main aim of the plant breeding are to improve yield, quality, disease resistance, and stress tolerance, Hybridization is practiced for outbreeding crops. The yield depends primarily on photosynthesis; Accordingly, photosynthetic processes are the prime targets of genetic engineering techniques. Much genetic engineering work is now being done on one of the most important enzymes, viz., ribulose bisphosphate carboxylase oxygenase (Rubisco). The genes for the active rubisco polypeptide from a photosynthetic bacterium can be cloned into Escherichia coli and they then synthesize larger amounts of the enzyme.
- The first intentional transfer of nif genes to an alien genetic background was the production by plasmid-mediated conjugation of nitrogen-fixing derivatives of Escherichia coli strains C-603. E.coli lacks nif genes and cannot fix nitrogen, but the acquisition of nif from Klebiella pneumoniae enables it to fix nitrogen. Later, similar transfers were achieved from Rhizobium trifolli to Kiebsiella aerogenes. The manipulation of nif genes into allen prokaryotic background is now a fairly routine process.allen procaryotic background is now a fairly routine process.