Nucleic acid extraction and purification principles and requirements
1. Ensure the integrity of the primary structure of nucleic acid;
2. Eliminate the contamination of other molecules (such as RNA interference when extracting DNA);
3. There are no organic solvents and high concentrations of metal ions that have inhibitory effects on enzymes in nucleic acid samples;
4. The contamination of other biological macromolecules such as proteins, polysaccharides and lipid molecules should be minimized;
5. Exclude the contamination of other nucleic acid molecules, such as RNA should be removed when extracting DNA molecules, and vice versa.
There are many methods for nucleic acid isolation and purification, and different schemes should be adopted according to the nature and initial amount of specific biological materials, and the nature and use of the nucleic acid to be isolated.
Regardless of the approach taken, there are general principles that should be followed:
One is to ensure the integrity of the nucleic acid primary structure because the complete primary structure is the most basic requirement for nucleic acid structure and function research;
The second is to try to exclude contamination from other molecules to ensure the purity of nucleic acid samples, which is the main content discussed in this chapter.
Preservation of Nucleic Acid Integrity
In order to ensure the integrity of the nucleic acid, during the operation, the damage to the nucleic acid by various harmful factors should be avoided as much as possible. Nucleic acid integrity is affected by many factors, including physical, chemical and biological factors, some of which can be avoided. For example, too acid or to alkali can destroy the phosphodiester bond in the nucleic acid chain. In the process of nucleic acid extraction, using a suitable buffer and always controlling the pH between 4 and 10 can be well avoided. In addition, as high-temperature heating, in addition to the destruction of chemical bonds in nucleic acid molecules by high temperature itself, may also bring liquid shear force due to boiling, so nucleic acid extraction is often carried out under the conditions of 0 to 4 °C.
Secondly, for unavoidable harmful factors, various measures should be taken to minimize the damage to nucleic acids caused by various harmful factors. The steps of separation and purification should be simplified as much as possible, the extraction time should be shortened, and the damage of nucleic acid integrity by various harmful factors should be reduced; the activation of DNase (DNase or DNAase) requires divalent metal ions such as Mg2+ and Ca2+. Salt and operation at low temperatures can basically inhibit the activity of DNase. The widespread existence and inactivation characteristics of RNase (RNase or RNAase) determine that biodegradation is the main hazard factor in the RNA extraction process.
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Nucleic acid extraction and purification principles and requirements…
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