Biotechnology is the use of artificial methods to modify the genetic material of living organisms or cells to produce novel compounds or to perform new functions.
To accomplish that biotechnologists must be able to extract, manipulate, and analyze nucleic acids.
Genomic DNA isolation steps
Most nucleic acid extraction techniques involve steps to break open the cell, and then the use of enzymatic reactions to destroy all undesired macromolecules.
| Steps to DNA Extraction | |
|---|---|
| 1. | Break the cells open to expose DNA |
| 2. | Remove membrane lipids by adding detergent |
| 3. |
Precipitate DNA with an alcohol - usually ethanol or isopropanol. Since DNA is insoluble in these alcohols, it will aggregate together, giving a pellet upon centrifugation. This step also removes alcohol-soluble salt. |
Cells are broken open using a detergent solution containing buffering compounds.
To prevent degradation and contamination, macromolecules such as proteins and RNA are inactivated using enzymes.
The DNA is then brought out of solution using alcohol.
The resulting DNA, because it is made up of long polymers, forms a gelatinous mass.
RNA is studied to understand gene expression patterns in cells.
RNA is naturally very unstable because enzymes that break down RNA are commonly present in nature.
Agarose gel electrophoresis for the separation of DNA fragments
Gel electrophoresis is a technique used to separate charged molecules on the basis of size and charge.
| DNA Gel Electrophoresis | |
|---|---|
| Separation method | |
| Principle: | |
| 1. | the movement of charged molecules in electric field |
| 2. |
the movement direction from - to + The nucleic acids consist of negatively charged phosphate groups. |
| 3. |
DNA-rate in gel depends on DNA-fragment length in indirect proportion The length of unknown fragments is compared to the length of standard fragments. |
Being acidic, DNA has a negative charge. Therefore, the fragments tend to move toward the gel’s positive end, with the smaller fragments moving more quickly.
After a period of time, the fragments separate into a pattern of bands.
This pattern is called a DNA fingerprint.
