The role of alcohol in DNA extraction is to precipitate DNA into a visible form. Also, it’s used in DNA washing and storing.
In this article, we will discuss the chemistry behind DNA precipitation. We’ll also discuss the role of water, alcohol and salt in DNA precipitation.
Let’s start the topics,
“Reaction between solute and solvent creates an insoluble solid substance, called as a precipitate and the process is referred to as precipitation.”
A very important step in genomics and genetics is DNA extraction. One of the crucial factor in any of the genetic process is the quality and quantity of DNA. It depends on the chemicals used in isolating DNA.
Alcohol is one of the essential ingredients of DNA extraction, used for precipitating, storing and washing DNA. Maniatis and Sambrook’s DNA extraction using the alcohol method is most popular DNA isolation method till date.
Read further on DNA extraction: Different types of DNA extraction/ isolation methods
Alcohol with some salt, precipitate DNA into a solid- visible form. Salts like Sodium acetate, sodium chloride and ammonium chloride are routinely utilised in DNA extraction.
A precipitated DNA appears as like a threat of cotton inside the tube. Due to the reaction between DNA, salt and alcohol it happens. However, the success of the precipitation depends on which DNA precipitation protocol you use.
For fulfilling the present purpose common alcohol- ethanol is widely used. Notably, other alcohols like methanol can also be used but it’s less effective.
Role of alcohol in DNA extraction:
DNA is soluble in water. Similar chemical structure of solute and solvent, dissolve properly in each other. Polar and non-polar molecules dissolve in polar solution and non-polar solution, respectively.
As we know,
DNA is a polar molecule with the net negative charge on the backbone of it (PO3–). For more detail on the structure of DNA read the article: DNA story: The structure and function 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.
With this, DNA is also hydrophilic in nature. Hydrophilic solutions easily dissolve in water because it attracts water (Hydro means water & philic means to attract). Hydrophobic solutions repel water and unable to dissolve in it.
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 the 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 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.”
Read further on dielectric constant: What is 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 to dissolve in alcohol is less as compared to the 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 adding alcohol.
Alcohol interacts with water by hydrogen bonding and makes the positive charge of water busy doing this. Hence alcohol protects the complex of PO3– and Na+ by neutralizing the charge of water.
Collectively, the DNA is pulled out from the bottom of the tube as a White cottony thread like precipitate. See the image below,
Sodium acetate is often used with ethanol in DNA precipitation, usually. As per my opinion, use isopropanol if less sample is available. It precipitates very effectively than ethanol.
Furthermore, alcohol is very useful in preserving DNA too.
It is a good DNA preservative because it’s non-reactive with DNA. here, the precipitates of DNA are collected in an alcohol-filled tube. Then it is stored as per your requirement (-4°C to -20°C for short-term storage and -194°C for long-term storage).
Precipitated DNA can’t directly applicable in downstream applications, although.
One of the important application of alcohol precipitation is that the alcohol-preserved DNA can dissolve properly (because the integrity of DNA is maintained).
Washing is as important as other steps in getting DNA, why?
During the isolation, proteins and traces of other chemicals remain with DNA. Once DNA precipitated, other debris is removed by high-speed centrifugation. Two or three times passing alcohol from DNA removes all other impurities besides DNA.
Impurities remain in the aqueous phase and removed by draining the DNA. Washing steps are repeated until clear pellets of DNA observed.
Now in the next step, precipitated DNA re-dissolved 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 or PCR.
In manual DNA extraction protocols like PCI, proteinase K or CTAB DNA extraction, precipitation step is very crucial. And hence the role of alcohol is unmatched.
Now, at last, I am giving you game-changing tips to improve the quality and quantity of DNA. Use chilled- alcohol for precipitation. However, the exact mechanism behind why chilled alcohol is so effective is still unknown. But believe me, it works!
Comment in below and let me know your experience with alcohol precipitation.
- Polymerase chain reaction
- DNA sequencing
- DNA microarray
- Fluorescence in situ hybridization
What is the use of alcohol in DNA extraction?
As we said, alcohol precipitates DNA into visible form like a cotton thread. it is also used for preserving and washing the DNA.
What is the function of 70% alcohol in DNA extraction?
The function of 70% alcohol is to precipitate DNA. Also, it’s applicable for storing DNA.
Add 700 ml of alcohol in 300ml of water which makes 1 litre 70% alcohol
What is the function of 100% alcohol?
The function of 100% alcohol is to wash the DNA. Other impurities of DNA removed by washing.
Why do we use chilled alcohol?
Chilled alcohol increases the yield of DNA.
What is the purpose of salt in DNA?
Salts interacts with the negative charge of DNA and increases the quantity of precipitate.