In this article we will discuss about the steps and applications of Recombinant DNA Technology.
Steps Involved in Recombinant DNA Technology:
The sequences of steps in r-DNA technology are as follows:
(i) Cell culture of cells with the required DNA sequences is obtained through tissue culture technique,
(ii) Specific restriction endonuclease is selected from the specific bacterium,
(iii) Restriction endonuclease cuts the specific DNA (desired gene) at the two ends which becomes the restriction fragment,
(iv) Same restriction endonuclease cuts a matching DNA sequence from a plasmid,
(v) Ligase enzyme which acts as a molecular glue joins the restriction fragment in the place vacated by the cut DNA segment of the plasmid. The plasmid DNA now contains foreign DNA and its DNA is called recombinant DNA (r-DNA),
(vi) Recombinant plasmids containing recombinant DNA are now inserted into a host cell, e.g., E. Coil. This is commonly done by a process called electroporation in which transient temporary pores are created in the cell membrane of the host for the uptake of foreign DNA molecules, and
(vii) Host bacterium divides to give multiple copies of the recombinant DNA which may be preserved in a DNA library which is also called genomic DNA library.
Genomic DNA Library:
A gene library contains several copies of many of the desired genes or DNA fragments. These may be present in bacterial clones with recombination DNA or by obtaining complementary DNA (c-DNA) by obtaining a DNA from an RNA template by using the enzyme reverse transcriptase. A collection of different c-DNA fragments is a c-DNA library. Gene libraries are maintained through special techniques.
Applications of Recombinant DNA Technology:
The technique of recombinant DNA can be used as follows:
1. It can be used to elucidate molecular events in the biological process of cellular differentiation and ageing.
2. This can also be utilised in making gene maps with precision.
3. This can be used to spell out the complete nucleotide sequence of genome of various organisms including humans.
4. By this technique, useful chemical compounds can be produced cheaply and efficiently.
5. Vaccines for hepatitis B have been produced by recombinant DNA technology.