Gene Cloning
- If a gene from humans is placed in bacteria, it does not produce any protein or multiply, as bacteria does not recognize the gene as a gene. For the recognition and multiplication of the gene it has to carry some identification sequences or replicons. Such replicons are known as vectors or cloning vehicles. A composite DNA molecule formed by joining a gene or insert with a cloning vehicle is called as recombinant DNA or chimeric DNA or chimeras. The process of creating a chimeric DNA is called as gene cloning, genetic engineering, gene manipulation or molecular cloning.
1. The first step in gene cloning is isolation of DNA from the organism being studied. Sometimes the DNA is made by using mRNA as reference or by chemical synthesis(PCR). Whatever be the method, the first step is to obtain the DNA molecule of interest.
2. The next step is to construct the chimeric DNA or recombinant DNA molecules by joining the vector or cloning vehicle with insert. Depending upon the host cell into which we transfer the insert, the vector or cloning vehicle varies. We use plasmids or phages for cloning in bacteria and SV 40 virus for cloning in animal cells.
3. The next step is transfer of recombinant molecules into the host so that it multiplies in the host. When the host cell divides, copies of the recombinant DNA molecules are passed on to the progeny.
4. The last step is identification of the bacteria which carries a recombinant molecule, from an array of bacteria with no recombinant molecules or having only a cloning vector. After these initial steps, the protocols or procedures will diverge depending upon the goal of researchers. If they want to study the sequence of DNA, they proceed with DNA sequencing. If they want to make the protein of the gene they proceed for gene expression. Whatever be the ultimate goal, the above said four steps remain constant and have to be carried out irrespective of whether the genetic engineer researcher s fresh or...