Explain the Outline of Recombinant Dna Technique

Explain the outline of recombinant DNA technique

Recombinant DNA refers to the creation of new combinations of DNA segments that are not found together in nature. The isolation and manipulation of genes allows for more precise genetic analysis as well as practical applications in medicine, agriculture, and industry.

The first step in making recombinant DNA is to isolating donor DNA. Isolation of donor DNA is accomplished by isolating cell. The method used to isolate is by disrupting lipid membrane with detergents, destroying protein with phenol or protease or by degrading RNAase. As a result, DNA leave the cell. Isolation of plasmid vector DNA is accomplished by an alkaline procedure or by boiling cell which removes bacterial chromosomal DNA from plasmid DNA. For the pure DNA, crude DNA is fractionated on a CsCl2 gradient, precipitated with ethanol or poured over a resin column.

Second step, that involved in recombinant DNA is cutting DNA. The DNA can be cut into large fragments by mechanical shearing or by using restriction enzyme. The function of enzyme is to recognize specific nucleotide sequence in the DNA and break the DNA chain at those points. These cuts will generate 'sticky or overhanging ends' or a blunt which generate flush ends.

Thirds step, joining DNA. After cut the donor DNA and vector DNA with restriction enzyme, they must be joined together. If both have been cut with same restriction enzyme, the ends will match up because they are sticky. DNA ligase are used to holds the ends of the DNAs together. It will create a phosphodiester bond between two DNA ends. DNA ligase refer as the glue of molecular genetics. After it is join together, called as recombinant DNA.

Fourth step, transformation of host. Recombinant DNA plus all other ligation product are mixed with host solution. Chemical are used to activate uptake by host. The host with vector or recombinant DNA is called Transformant then host become antibiotic resistant. Transformation result are some hosts will not uptake anything, some will uptake vector alone or some will take recombinant DNA. Fifth step, growth of transformant. Transformants and other products are cultured in a dish containing nutrients, antibiotic and colorless dye called X-galactose. Recombinant plasmid DNA will form colonies on the dish while, recombinant viral DNA will form plaque.

Sixth steps, selection for recombinant. X-galactose in nutrient dish allows selection of successful ligation of DNA that inactivation of LacZ gene and white colour. Ampicillin in nutrient allows selection of successful transformation. Finanly, analysis of recombinant DNA technology. Colonies that produce B-galactosidase and are fed x-gal will turn blue. On the other hand, colonies that do not produce B-galactosidase remain white in colour, even in the presence of xgal. Therefore, untransformed bacteria indicate