As one of the pathological diagnosis techniques, molecular pathological diagnosis applies molecular biology to detect molecular genetic changes in cells and tissues at the genetic level. With these results, it serves to coordinate the pathological diagnosis and genotyping, guide the targeted therapy, predict the therapeutic response, and evaluate the prognosis.

Genes carry the genetic information for the organism. Genetic testing may help predict the risk of a particular disease and enable early, effective prevention and intervention. A gene, the basic unit of heredity, is a segment of DNA necessary to synthesize a polypeptide or functional RNAs. To make it easy to understand, one may simply regard it as a special fragment in the long DNA strand.

To obtain nucleic acid is the first step towards good genetic research. Nucleic acid extraction is the common first step to kickstart biological research while its quality is also crucial to the success of subsequent experiments. The performance of processes including cloning, PCR, QPCR and library construction for sequencing all require nucleic acids. Today, let us take a quick look at the basic principles and methods of nucleic acid extractions.

What is Nucleic Acid?

Nucleic acids come in two forms—deoxyribonucleic acid (DNA) and ribonucleic acid (RNA); DNA is mostly concentrated in the nucleus, mitochondria and chloroplasts, while RNA is mostly in the cytoplasm.
Providing the basic materials for gene expression, nucleic acids are the main object of research in molecular biology. No matter in structural or functional studies of nucleic acids, the first step is always extraction and purification. Nucleic acid extraction is fundamental to a vast array of studies and applications.

Basic Steps of Nucleic Acid Extraction

1. Lysing the cell to release nucleic acids. Lysis buffer is used to break down the cell structure of the sample, thus freeing DNA from the sample into the lysis system;
2. Isolating and purifying nucleic acids. Proteins bound to nucleic acids as well as biomacromolecules such as polysaccharides, lipids and other unwanted nucleic acid molecules should be removed;
3. Concentrating and precipitating nucleic acids;
4. Purifying nucleic acids. Purification is the process that completely separates DNA from other components in the lysis system, such as proteins, salts and other impurities.

Principles and Requirements of Nucleic Acid Extraction and Purification

1. The primary structure of the nucleic acid should be kept integral to prepare for the subsequent experiments;
2. Contamination by other nucleic acid molecules should be excluded (avoiding interference from RNA when extracting DNA and vice versa);
3. Enzyme-inhibiting organic solvents and high concentrations of metal ions should be absent in the nucleic acid samples;
4. Contamination of nucleic acid samples with other biomacromolecules such as proteins, polysaccharides and lipid molecules should be minimized.

Common Methods of Nucleic Acid Extraction and Purification

(1) According to the approach adopted in nucleic acid extraction, the methods can be divided into manual extraction and high-throughput automated extraction.

(2) According to the extraction principle, the methods can be categorized as follows:

1. Boiling lysis method: this method is generally used for manual extraction of DNA. The chromosomal DNA is much larger in size compared with the plasmid DNA. The former is a thread-like molecule, while the latter is a covalently closed circular molecule. When the DNA solution is heated, the thread-like chromosomal DNA is easily denatured while the covalently closed plasmid DNA regains its natural conformation upon cooling. The denatured chromosomal DNA fragments precipitate, bound to the denatured proteins and cellular debris, while the renatured supercoiled plasmid DNA molecules stay in solution in the liquid phase. This allows them to be separated via centrifugation.
   DNA extraction method by boiling lysis, with low yield, quite a few impurities and probability of DNA breaks, is mostly applied in rough experiments.
2. Phenol-chloroform extraction: this is a classic method for DNA extraction. It separates and removes the protein using two different organic solvents alternately. When the homogenate is treated with phenol, the bond between protein and DNA is broken. As the protein molecules, with the large number of polar groups on their surfaces, are soluble to phenol and as phenol inhibits the degradation of DNase, protein molecules are solubilized in the phenolic phase while DNA is solubilized in the aqueous phase. After centrifugation, the aqueous layer is removed, which is repeated for several times before pooling together the DNA-containing aqueous phase. Considering the insolubility of nucleic acids in alcohol, ethanol is used to precipitate DNA. After centrifugation, DNA can be harvested.
   The greatest advantage of phenol-chloroform extraction is its low cost and low demand on the experiment conditions.
   The extracted DNA remains in a nature status with high purity, long fragments and high quality,but the complexity of operation process cannot be regardless at the same time.
3. Salting-out method: as a variant of the phenol-chloroform extraction, this method separates RNP and DNP by taking advantage of their different solubility in the electrolytic solution. As the strength of this method, it overcomes the operational troubles and almost all other disadvantages of phenol chloroform extraction. However, the purity of the extracted DNA is not stable.
4. Anionic detergent method: once proteins are denatured with detergents such as SDS or sodium xylene sulfonate, DNA can be extracted directly from biological materials. Since DNA and proteins in cells are often bound by electrostatic forces or coordinate bonds, anionic decontaminants being able to break these bonds are commonly used to extract DNA.
   This SDS method has simple and gentle operation processes and yields high molecular weight DNA. However, it will generate more carbohydrate impurities in its product.
5. Guanidine isothiocyanate/phenol method (Trizol method): It is a classic method for RNA extraction. In homogenized or lysed samples, Trizol maintains the integrity of the RNA while destroying cells and lysing cellular components. After centrifugation with chloroform, the lysate is stratified into an aqueous phase and an organic phase. RNA is present in the aqueous phase. After the aqueous phase is separated, isopropanol is used to precipitate and recover RNA. When the aqueous phase is removed, DNA can be harvested in the intermediate phase with ethanol precipitation and protein can be obtained from the organic phase with isopropanol precipitation.
   The Trizol method is suitable for RNA extractions from common plant tissues, animal tissues, as well as fungi and bacteria.
6. Principle of CTAB method (a classical method for plant DNA extraction): CTAB (hexadecyltrimethylammonium bromide), a cationic detergent, is capable of precipitating nucleic acids and acidic polysaccharides from low ionic strength solutions. CTAB forms complexes with proteins and polysaccharides in high ionic strength solutions (>0.7 mol/L NaCl) but would not precipitate nucleic acids. Once impurities such as proteins, polysaccharides and phenols are removed by organic solvents, nucleic acids can be isolated with ethanol precipitation.
7. Spin column-based purification: nucleic acids are separated and purified by special silica-based adsorbent materials that can specifically adsorb DNA while allow RNA and protein to pass through. Solution with high salt concentration and low pH is used to bind nucleic acids and that with low salt concentration and high pH is used for elution to isolate and purify nucleic acids. Spin-column purification is also commonly used in extraction kits.
   The spin column DNA extraction kit is less expensive, easier to operate and is more widely used commercially. However, it causes more loss and requires larger quantity of samples, therefore it’s hard to operate when handling with valuable rare samples. At the same time, it is inconvenient for high-throughput and automated operations.
8. Magnetic bead-based extraction: This method follows the principle that the active group of magnetic particles can absorb and desorb nucleic acids under certain conditions. After the cells lysed by lysis buffer, the magnetic particles with active groups can specifically bind to the nucleic acid molecules freed from the cells while other interfering substances in the sample can be well removed. Located in the magnetic field, the magnetic particles are separated from the liquid and later recovered (in the form of magnetic bead-DNA mixture) for elution to obtain purified DNA and nucleic acid templates of high quality.

The magnetic bead-based method does not require centrifugation, the addition of multiple reagents and is simple to operate, which meets the requirements of automated nucleic acid extraction. However, it is difficult to use this method universally in research due to its high cost.

There is a famous saying that “a workman must first sharpen his tools if he is to do his work well”. To accelerate the extractions and purifications of nucleic acids and proteins, high speed microcentrifuge is a must-have. With a speed of up to 15,800rpm, RWD M1324/M1324R Microcentrifuge make your centrifugation simple. I believe you should all have a general understanding of the basic nucleic acid extraction methods.