Wednesday, August 5, 2009

How do we Manipulate Genes?

Before beginning any genetic engineering project, it is necessary to obtain a reasonable amount of relatively pure DNA, which is then cut and ligated to build the new gene. Today, several companies make DNA extraction and purification kits, making the genetic engineering process much simpler. The basic procedure requires the releasing of the DNA from the cells and purification of the DNA to be used in the experiments.

In a typical extraction and purification procedure for plasmid DNA, bacterial cells with the desired plasmid are lysed (broken up) under alkaline conditions and the crude lysate (remains of the cells) is purified using either filters or centrifugation. The lysate is then loaded onto an apparatus where plasmid DNA selectively binds under appropriate low-salt and pH conditions. RNA, proteins, metabolites, and other low-molecular-weight impurities are removed by a medium-salt wash, then plasmid DNA is released in high-salt buffer. The DNA can then be concentrated and desalted for genetic engineering uses.

General Steps for Gene Manipulation are as under:
1) Grow the bacteria in liquid culture.
2) Centrifuge the bacterial suspension to concentrate the bacteria.
3) Discard the supernatant (the liquid part that remains above the pellet).
4) Resuspend the bacteria pellet in a solution with RNAse (enzyme that degrades RNA).
5) Add a buffer to promote an alkaline lyse of the bacteria.
6) Neutralize and adjust the saline conditions of the suspension with the buffer.
7) Centrifuge to separate proteins and other impurities.
8) Adsorb the plasmid DNA onto a membrane by filtration.
9) Rinse the membrane with a solution containing ethanol.
10) Elute plasmid DNA from the membrane with EDTA, a chemical substance that preserves the integrity of DNA.

Tags: Bio Technology, Bio Genetics, Genetic Engineering

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