In this article we will discuss about the Recombinant DNA Technology:- 1. Introduction to Recombinant DNA Technology 2. Biomedical Importance of Recombinant DNA Technology 3. Concept 4. Some Practical Applications.
Introduction to Recombinant DNA Technology:
a. Recombinant DNA technology is better referred to as genetic engineering.
b. Much has been learned about the diseases from the study of affected proteins, but this mechanism cannot be applied where the specific genetic defect is unknown. This new technology overcoming these limitations will approach directly to the DNA molecule for information.
Biomedical Importance of Recombinant DNA Technology:
a. It is helpful to give clear idea regarding the molecular basis of a number of diseases (e.g., familial hypercholesterolemia, sickle cell disease, the thalassemia, cystic fibrosis, Huntington’s chorea).
b. Using this technology, a large quantity of human proteins can be produced for therapy.
c. By its aid proteins for vaccines (e.g., hepatitis B) and for diagnostic tests (e.g., AIDS test) can be obtained.
d. This technology is utilized to diagnose existing diseases and predict the risk of developing a given disease.
e. Gene therapy for sickle cell disease, the thalassemia’s, adenosine deaminase deficiency, and other diseases may be devised.
Concept used in Recombinant DNA Technology:
Isolation and manipulation of DNA is the object of recombinant DNA research. This requires several techniques and reagents.
a. Some endonucleases that cut DNA at specific DNA sequences within the molecule are a key tool in recombinant DNA research. These enzymes were originally said to be restriction enzymes. More than 200 defensive enzymes protect the host bacterial DNA from foreign organism (primarily infective phages).
They are only present in cells that also have a companion enzyme that methylate’s the host DNA giving it an unsuitable substrate for digestion by the restriction enzyme.
b. The restriction enzymes are named vide the bacterium from which they are isolated (e.g., Eco RI from Escherichia Coli, Bam HI from bacillus amyloliquefaciens).
c. Each enzyme recognizes and cleaves a specific double-stranded DNA sequence. These DNA cut result in blunt ends or overlapping (sticky) ends (Bam HI) (Fig. 24.1), depending on the mechanism used by the enzyme. Sticky ends are particularly useful in constructing hybrid or chimeric DNA molecules.
d. In case the nucleotides are distributed randomly in a given DNA molecule, one can easily calculate how frequently a given enzyme could cut a length of DNA.
e. For each position in the DNA molecule there are 4 possibilities (A,C, G or T); therefore, a restriction enzyme that recognizes a 4-bp sequence will cut, on average, once every (44), whereas another enzyme that recognizes a 6-bp sequence will cut once every (46).
f. When DNA is digested with a given enzyme, the ends of all the fragments will have the same DNA sequence. The fragments produced can be isolated by electrophoresis.
Preparation of Chimeric DNA Molecules:
a. Sticky ends of a vector may reconnect with themselves with no gain of DNA. These ends of fragments can also anneal so that tandem heterogeneous inserts form. These end sites may not be available or in a convenient position.
b. To overcome the above problems, an enzyme that generates blunt ends is used and new ends are added using the enzyme terminal transferase.
c. If poly d(G) is added to the 3′ ends of the vector and poly d(C) is added to the 3′ ends of the foreign DNA, the two molecules can only anneal to each other and thus overcome the above problem. This procedure, called homopolymer tailing, also generates an Sma I restriction site.
d. Sometimes, synthetic oligonucleotide linkers with a convenient restriction enzyme sequence are ligated to the blunt- ended DNA. This technique has the advantage of joining together any pairs of ends. The dis-advantages are that there is no control over the orientation of insertion of the number of molecules annealed together.
Some Practical Applications on Recombinant DNA Technology:
a. Specific genes to distinct chromosomes are localized by this technique and thus to define a map of the human genome. This is already producing useful information in the definition of human disease.
b. Somatic cell hybridization and in situ hybridization are two techniques used to accomplish this.
c. In hybridization, the simpler and more direct procedure, a radioactive probe is added to a metaphase spread of chromosomes on a glass slide. The exact area of hybridization is localized by layering photographic emulsion over the slide and, after exposure lining up the grains with some histologic identification of the chromosome. Some of the human genes are localized by this technique.
d. Genes that code for proteins with similar functions can be located on separate chromosomes.
e. Genes that form part of a family can also be on separate chromosomes (Growth hormone and prolactin).
f. The genes involved in many hereditary disorders known to be due to specific protein deficiencies, including X chromosome-linked conditions, are really located at specific sites.
a. This technology has two prominent merits:
(i) It can supply large amounts of materials that could not be obtained by conventional purification methods.
(ii) It can provide human material (e.g., insulin, growth hormone).
b. Although the primary aim is to supply products, generally proteins, for treatment (Insulin) and diagnosis (AIDS test) of human and other animal disease and for disease prevention (hepatitis B vaccine), there are other real and potential commercial applications, especially in agriculture.