Recombinant DNA Technology is the process that isolation and manipulation of DNA sequences. it involves cutting, combining of DNA from different sources and create new DNA combination/genes.
Gene Cloning
Gene cloning is the process of generation of genetically identical copy of cell or an organism.
A fragment of DNA, that gene of interest on cloning is inserted into a circular DNA molecule called vector, and this vector DNA molecule is called recombinant DNA molecules and vector is called recombinant vector. and this methodology is recombinant DNA technology.
Within the host cell recombinant vector multiples and produce identical copies, and when the host cell divided recombinant vector are passed to the next progeny, and further development of identical copies.
The cells that contains recombinant vectors are identified by marker assistance and specific traits that are determined by recombinant vector gene (antibiotic resistance trait)
Steps of Gene cloning
- Purification of DNA from living cells – lsolate the plasmid DNA, isolate target DNA
- Manipulation of the purified DNA – cut the plasmid using restriction enzymes, cut the target DNA segment using same restriction enzyme, combine the target DNA and plasmid.
- Introduction of DNA into Living Cells/vector – recombinant plasmids will be also multiplied inside bacteria, select for host cells carrying the recombinant plasmid using specific traits that are determined by recombinant vector gene (antibiotic resistance trait)
Genetic Engineering
Genetic Engineering is the process of direct manipulation of organism’s genome, by introducing desired gene which is responsible for desirable traits using recombinant DNA technology.
Benefits of the recombinant DNA technology
- Development of transgenic plants – pest, diseases resistance, abiotic stress resistance plant varieties
- Development of transgenic animals – Mice (for research to study human immune system), chickens( resistance to infections), cows (give more meat and milk), goats, sheep (produce human proteins in their milk)
- Recombinant Bacteria used as bioreactors to produce useful enzymes such as human insulin, growth hormones, and industrial useful enzymes.
- Recombinant Bacteria can be used for eat oil spills.
DNA manipulative enzymes using recombinent DNA technology.
- Nucleases
- Ligases
- Polymerases
- Modified enzymes
Nucleases
- Breaking the phosphodiester bonds
Two different types
- Exonucleases – remove nucleotides one at a time from the end of the Nucleotides.
- Endonucleases – Break the internal phosphodiester bonds.
Discovery of the restriction endonucleases
Restriction endonucleases naturally occur in bacteria as a defense mechanism. Some bacteria strains are shown immunity to bacteriophage (viruses that infect the bacteria). Restriction endonucleases degrades the page DNA therefore these genes are immune to the bacteriophage attacks.
However these restriction enzyme can not degrade its own DNA because of the methyl groups blocks the recognition sites for the restriction endonucleases.
Three different classes of restriction endonucleases are recognized, each distinguished by a slightly different mode of action.
- Types I and III only a limited role in genetic engineering
- Type II is important in gene cloning
Restriction endonucleases – Nomenclature
Naming system based on bacterial genus, species,strain and isolation.
- Bam HI Bacillus amyloliquefaciens strain H, isolate I
- Hind III Haemophilus influenzae strain Rd, isolate II
Characteristics of restriction sites
- Palindromic – two fold symmetry or inverted repeats.
- Cutting leaves 5’ or 3’ overhangs or blunt ends
Cutting frequency of the restriction enzyme recognition sites
When shorter the recognition site sequence, cutting frequency will be increased.
- Cutting frequency will be proportional to the cutting 4n (n = number of nucleotide of the restriction sites)
Characteristics of restriction enzymes
- Ambiguous – One enzyme recognized more than one DNA sequence as its target site. it is important to avoid non specific cleavages and false positive results, however it is used full when the target site is unknown
- Some restriction sites recognize the different restriction enzymes
- Same cohesive end can be forms by different restriction enzymes
Ligases
Join the single stranded breaks and join two individual DNA fragments by forming phosphodiester bond between the 5’ phosphate group and the 3’ OH group of the two ends.
DNA-modifying enzymes
Alkaline Phosphatase – Removes the phosphate group present at the 5′ terminus of a DNA molecule.
Polynucleotide kinase – Reverse effect to alkaline phosphatase.
Terminal deoxynucleotidyl transferase – adds one or more deoxyribonucleotides onto the 3′ terminus of a DNA molecule