Different methods of nucleic acid purification
Different types of nucleic acid purification methods are available for different types of tissue. Alcohol purification, silica gel column based purification, agarose gel-based DNA purification, automated nucleic acid purification and chemical based nucleic acid purification methods are commonly used in genetic laboratories.
In fact, the nucleic acid purification is followed by the extraction method or both methods are combined with each.
For understanding the nucleic acid purification we have to first understand several terminologies such as nucleic acid, extraction or isolation and purification of nucleic acid.
The nucleic acid is the organic acid made up of sugar, phosphate and nitrogenous bases. Based on the sugar molecules present into the nucleic acid, it is categorised into deoxyribonucleic acid or ribonucleic acid (called as DNA or RNA respectively).
The DNA is the basic unit of inheritance; made up of deoxy sugar, phosphate and nitrogenous bases ( purines and pyrimidines).
Instead of thymine in DNA, the uracil is present in RNA which is the basic and the main difference between DNA and RNA.
Read further on DNA: DNA story: The structure and function of DNA
Isolation, extraction and purification
Actually, there is no major difference between isolation and extraction, However, the minor difference both the terms is as explained,
Extraction is one part of the whole process of isolation. Isolation means isolation something, isolating DNA from other biomolecules for that purpose we have to first extract DNA from the cell.
In general, the isolation method is a combination of extraction + purification
Purification is another method followed by the extraction. There are many impurities present in the extracted biomolecule. For example, if we have to isolate DNA, we have to first extract DNA from the cell followed by the purification of DNA from the other cell debris.
Depending upon the type of the extraction sample the protocol of the extraction varies. Human DNA purification, plant DNA purification, viral DNA purification and bacterial DNA purification each DNA purification method is different from each other.
Importance of nucleic acid purification:
The highly purified nucleic acid increases the yield of the experiment. Failure in the downstream experiments is due to the contaminated sample.
The chance of a false positive result is very high in the unpurified nucleic acid.
Contamination decreases the specificity of the reaction also the impurities present into the nucleic acid hinders into the reaction and slow down the reaction.
For more detail on the protein and RNA contamination into the DNA sample please read the article: A complete guide for analysing and interpreting gel electrophoresis results
Alcohol DNA purification system:
Alcohol is used in DNA purification since long. The Sambrook’s DNA extraction protocol is entirely based on the alcohol purification. In other words, we can say that the alcohol purification step is the sub-step of any DNA extraction protocol.
Methanol, ethanol and isopropanol are some of the common alcohol used in nucleic acid purification. The alcohols used in DNA as well as RNA purification.
Once the DNA/ RNA is precipitated, it is washed using alcohol. Generally, 1 ml of alcohol is enough for the washing.
After addition of the alcohol, the mixture is vortexed gently or inverted several times until the clear DNA/ RNA pellet observed.
After that, the sample proceeds for high-speed centrifugation. Here, due to the centrifugal force, the debris and other contaminant separated from the nucleic acid.
The contaminants are removed by removing the alcohol. 3 alcohol washes are enough for getting pure nucleic acid.
For more detail on the role of alcohol in DNA extraction read the article: Role of alcohol in DNA extraction
This method is simple, effective, rapid and impactful. Alcohol nucleic acid purification is used in phenol-chloroform DNA extraction method, proteinase K DNA extraction method and other salt-based DNA extraction methods.
Read further: Different types of DNA extraction methods
Spin column-based nucleic acid purification
The column- based nucleic acid purification is one of the most advanced, accurate and rapid methods of DNA purification, also called as a solid phase nucleic acid purification.
The method is entirely based on the centrifugation (spin), the silica is used as a solid phase which is settled inside the column tube. The spin column tube is made up of three major parts:
The silica as a solid phase into the column, the column and the collection tube.
The chaotropic salts are the major constituent of the spin column buffers. The chemistry of the spin column method is based on the pH and the salt concentration. However, the composition and the types of salt used into the spin column are never disclosed by the manufacturer.
The buffer of the spin column technique is made up of the alcohol and salts. Once the desired pH is achieved, the DNA molecules bind with the silica of the column. The DNA can not pass through the column.
Under the high-speed centrifugation, the salts and alcohol wash the DNA and removes other cell debris from the DNA.
The DNA remains bound to the silica and the other impurities are collected into the collection tube. The graphical representation of the spin column based nucleic acid purification method is given into the figure below.
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The elution buffer or the TE buffer contains the higher pH than the extraction buffer. Once we add the elution buffer to the spin column, DNA removed from the silica and dissolve into the buffer.
When we centrifuge the tube, the dissolved DNA detached from the silica and collected into the collection tube.
This technique is easy, reliable, less time consuming and effective. The yield of the result is also high.
Automated Nucleic acid purification method:
In present-day science, robotic techniques are more rapid and easy to perform. Automated machines for quantification and extraction are more effective and accurate than the manual methods.
The automated nucleic acid purification systems are based on the automation technology which utilises less human help. kingFisher series of purification machines by Thermofisher, MPure-12 by MP Biomedicals, Maxwell nucleic acid purification system by Promega and QIAube series of nucleic acid purification systems by Qiagen are some of leading automated nucleic acid purification systems available.
Thermofisher: KingFisher series of nucleic acid purification systems
Each technology has a different mechanism of working, However, majorly the automated nucleic acid purification technology is based on the magnetic bead technology.
Nucleic acid purification and yield analysis:
Quality and quantity of DNA is a very crucial parameter in DNA sequencing and microarray. DNA with 260/280 absorbance ration of ~1.8 and 50ng DNA is advisable for the downstream experiments.
For more detail on DNA quantification please read the article: Review of Qiagen QIAxpert
We can also perform simple agarose gel electrophoresis of gDNA for checking the purity of DNA. For more detail on agarose gel electrophoresis read the series of articles:
Alcohol is one of the important chemicals in nucleic acid purification. Each and every method of nucleic acid purification is based on the chemistry between DNA and alcohol. We can also purify DNA by agarose gel electrophoresis. If you know the technique please comment below and let us know.