“Reaction between solute and solvent creates an insoluble solid substance, called as a precipitate and the process is termed as precipitation.”
DNA extraction is a very important step in genomic research and diagnosis. Purity and quantity of DNA always matter while performing any downstream applications. The purity and quantity of DNA depend on the chemical used in the extraction.
Alcohol is one of the important ingredients in DNA extraction. It is used in precipitation, washing and storing DNA. Maniatis and Sambrook’s DNA extraction using alcohol method is one of the popular methods of DNA extraction till date.
An alcohol along with the salt, precipitate DNA into solid form. Sodium acetate, sodium chloride and ammonium chloride are most routinely used salts in DNA extraction.
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A precipitated DNA appears as like a threat of cotton inside the tube which is results from the chemical reaction between DNA, salt and alcohol. Commonly used alcohols for precipitation are ethanol, isopropanol and methanol.
Ethanol is widely applicable for precipitation and washing of DNA, while methanol is not a good choice, but it can work if we have no other option.
DNA is most soluble in water. Similar chemical structure of solute and solvent, dissolve properly in each other, polar solution can dissolve in polar and non-poplar solution can dissolve in non-polar solution.
DNA is a polar molecule with the net negative charge on the backbone of DNA (PO3–). For more detail on the structure of DNA read the article: Story of DNA
Water is also a polar solution with partial negative charge near the oxygen atom and partial positive charge near the hydrogen atom. So polar molecules like DNA can easily interact with water. electrostatically, free H+ of water and PO3– of DNA react with each other and makes DNA soluble in water.
Additionally, DNA is hydrophilic in nature. Hydrophilic solutions are easily dissolved in water because it attracts water (Hydro means water & philic means to attract). Hydrophobic solutions repel water and unable to dissolve in water.
In DNA precipitation, a salt (sodium acetate) reacts with DNA, it breaks up into Na+ and (CH3COO)–, the positively charged sodium ion neutralize negatively charged PO3– of DNA. Hydrophilic nature of DNA helps it to dissolve it in water but by reacting with sodium acetate, DNA becomes less hydrophilic.
Here the role of alcohol is become very crucial for getting good quality precipitate of DNA. According to the coulombs low, “force of attraction between two opposite charges is inversely proportional to the dielectric constant.”
Water has a high dielectric constant as compared to alcohol hence it hinders in Na+ and PO3– binding. On the other hand, alcohol has a lower dielectric constant. So it protects Na+ and PO3– complex from the water.
The dielectric constant of water is ~80 while ethanol is ~24. So the chance of DNA dissolve in alcohol is less as compared to water.
Ethanol has both polar and non-polar group. It has a non-polar C2H5 and a polar OH– group. Conclusively, as per the coulombs law, the force of interaction between PO3– and Na+ increased, after the addition of alcohol.
Alcohol interacts with water by hydrogen bonding and makes the positive charge of water busy doing this. Hence alcohol protects the interaction of PO3– and Na+ complex by neutralizing the charge of water. Collectively, DNA is pulled out on the bottom of the tube as a White cottony thread like precipitate.
Generally, ethanol is frequently used in precipitation with a combination of sodium acetate. But if the quantity of the sample is very less than isopropanol is the best option.
Furthermore, alcohol is very useful in preserving DNA. Having non-reactive with DNA, alcohol works as a preservative. Precipitated DNA is collected into alcohol containing tube and deep froze depending upon the type of storage requirement (-4°C to -20°C for short-term storage and -194°C for long-term storage).
Though precipitated forms of DNA is not useful for downstream practices, it is further very essential to dissolve DNA once again, after precipitation.
Dissolved DNA contains many impurities. The protein debris has still remained with DNA. after addition of alcohol DNA remain is precipitated form but other proteins become dissolved in homogenate. Alcohol can wash DNA by passing through DNA molecules and removes all impurities from DNA.
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The impurities collected into the aqueous phase of alcohol and removed by draining. The process is repeated until clear pellet of DNA is not observed. Precipitated DNA is re-dissolve in high pH solutions like TE buffer.
TE buffer has a higher pH than DNA so it dissolves DNA properly. Pure DNA solution is now ready for blotting and PCR.
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Article covered By- Ravi Parmar
Reviewed by- Tushar Chauhan