AN INTRODUCTION TO GENE CLONING!!
Hello everyone, As per schedule the cited blog for this month is GENE CLONING. We read about "Clone" in the past. A Clone is a group of identical cells inherited from a single mother. Here we produce a number of desired genes. Moving back, prior to gene cloning, the ability of a bacterial cell to take up the foreign DNA and getting incorporated right into the bacterial chromosome - A process known as TRANSFORMATION paved a better understanding of DNA as a genetic material. The main criteria is that DNA should come from the same species inorder to get integrated and passed on to daughter cells. Just like we can't join a chimpanzee DNA with some other mammal. This is because, the two strands should be compatible and complementary to each other. Theories talk but practicals prove!!! It is easier that we study the entire process as * Selection and cutting of foreign DNA * Insertion into the bacterial chromosome * Return of the cell to live conditions. But in practice it is highly inconceivable because the chromosome has got the following properties: * Far too large * Cannot be isolated intact nor handled easily in the test tubes * Returning of a cell is difficult Therefore two suitable classes of vectors or replicons are used. Namely, ~ Plasmids ~ Phages The entire steps in Gene Cloning is given below: 1. Cutting of DNA strands using Restriction Endonucleases: The restriction enzymes have a prominent practical application. They cleave the DNA fragments at "specific sequences" so that the two strands 'll be complementary to each other. Eg: EcoRI cuts the DNA strand between 'G' and 'T' producing single stranded tails of 4-8 base pairs length. 2. Joining the DNA fragments: Now that we have the two strands next method is joining the DNA fragment to a suitable vector. DNA ligase enzyme plays a major role by forming Phospho diester bonds between the strands thereby producing a complete Covalently Closed Circles (CCC) - Recombinant Cell. 3. Transformation: The next is the introduction of the Recombinant Cell into the host cell. Most of the cells resists this process as they lack the natural ability to uptake the foreign DNA. Stress conditions are hence applied. 4. Selection of Recombinants: The recombinants are selected using 1. Genetic methods 2. Immunochemical methods 3. Nucleic acid hybridization 5. Analysis of cloned DNA: The cloned DNA is finally analyzed using Hybridization Techniques and DNA sequencing. THIS BLOG IS FREE OF PLAGIARISM Reference: An Introduction to Recombinant DNA Technology, Chapter #2, Principles of Gene Manipulation - An Introduction to Genetic Engineering, RW Old, S B Primrose 5th Edition, BlackWell Science Unit, Page Nos: 23-25.