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Hot Shot Method of DNA Extraction
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Objectives

 

1.    To isolate DNA from fish fins.
2.    To obtain PCR-quality DNA from the fish fins.

 

Theory

DNA, the building block of life is the genetic material for all living organisms and it contains information that is crucial for heredity. DNA isolation is necessary for genetic analysis including scientific, medical, or forensic purposes. Presence of  lipids, proteins, polysaccharides and some other impurities in the DNA preparation can interfere with DNA analysis methods by reducing the quality of DNA,  which leads to its shorter storage life. DNA can be isolated from any living or dead organism. Commonly used sources for DNA isolation are whole blood, hair, sperm, bones, nails, tissues, saliva,  epithelial cells, urine,  bacteria, animal tissues, and plants.

 

The traditional methods for the isolation of DNA are more time consuming and the reagents used are costly. A few modifications in this protocol make it simple to prepare PCR-quality genomic DNA than traditional methods. In this modified method (HotSHOT method), samples are incubated briefly in hot NaOH and then neutralized by Tris buffer. This method found to be rapid, reliable, and inexpensive for the isolation of PCR-quality DNA from zebra fish tissues. These advantages make it useful for high-through put applications. Although it is a very rapid and simple method, the quality of DNA is not adequate for all applications. It is found that DNA obtained from this method is sheared and its concentration is very less. The DNA is extensively sheared and its concentration is too low. It is also possible that the DNA was not efficiently digested by the restriction enzyme because of interfering substances in the DNA preparation or because the DNA did not reanneal efficiently after it was neutralized. So this method is mainly for PCR related applications. Although HotSHOT DNA preparation is likely to be limited to PCR applications, the savings in time and reagents over traditional DNA preparation methods is substantial, and the quality of the results is as good as or slightly better than DNA prepared by traditional methods.

 

Principle

 

The isolation of DNA usually begins with lysis of tissue or cells which is essential for the destruction of protein structures and also allows the release of nucleic acids from the nucleus. Sodium hydroxide is mostly used to extract DNA out of a cell. It helps to break down the cell wall by loosening the hard structure of a cell wall or membrane. More importantly, NaOH disrupts the hydrogen bonding between the Nitrogen bases and  converting the double-stranded DNA (dsDNA) (including the genomic DNA (gDNA) and the plasmid) to single stranded DNA (ssDNA). This process is called denaturation and is the central part of this DNA extraction procedure. Presence of sodium hydroxide makes the solution very basic or alkaline. Tris-Cl maintains the pH of the solution. Basically it reacts with the lipopolysaccharides present on the outer membrane which make the membrane permeable

 



 

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