Processes of recombinant DNA technology

Here is the whole process of recombinant DNA technology, step by step.

1. Isolate the DNA. The DNA is inside membranes, mixed with other molecules. The cell is broken open with an enzyme that suits it: lysozyme for bacteria, cellulase for plant cells, chitinase for fungi. Enzymes then remove the rest: ribonuclease destroys RNA, protease destroys proteins. Adding chilled ethanol makes the purified DNA precipitate, as fine threads that can be spooled out.

2. Cut it. The DNA is cut with restriction enzymes, and the result checked by agarose gel electrophoresis. The vector is cut with the same enzyme, and the gene is joined to it with DNA ligase, giving the recombinant DNA.

3. Amplify the gene by PCR. The polymerase chain reaction makes many copies of a gene in a test tube, without living cells. Each cycle has three steps:

• Denaturation: heating separates the two strands of the DNA.
• Annealing: two short pieces of DNA, the primers, bind to the strands on either side of the gene.
• Extension: DNA polymerase builds new strands from the primers.

Repeating the cycle doubles the DNA each time, giving about a billion copies. The polymerase must survive the repeated heating, so a thermostable enzyme is used, Taq polymerase, from the hot-spring bacterium Thermus aquaticus.

4. Insert the recombinant DNA into host cells, and select the transformed ones, for example on a plate with ampicillin, if the vector carries the ampicillin-resistance gene.

5. Obtain the product. If the gene is expressed in a host that does not normally have it (a heterologous host), the protein it makes is called a recombinant protein. Small cultures give too little product, so cells are grown in large bioreactors (100-1000 litres), which provide the best conditions: temperature, pH, nutrients, oxygen. The most common is the stirred-tank bioreactor, where a stirrer mixes the contents and a sparger bubbles in sterile air.

6. Downstream processing. The product must be separated and purified, then formulated with preservatives. For medicines, it must pass clinical trials and strict quality control before it can be sold.