Talking about one of the extraction and purification of nucleic acids: design and principle of nucleic acid separation and purification
July 08, 2021
Section 1 Design and Principle of Nucleic Acid Separation and Purification
Nucleic acids in cells include DNA and RNA molecules, which combine with proteins to form nucleoproteins. DNA, protein and DNP&40; DNP and #41; RNA and protein binding to ribonucleoprotein 40; RNP and #41; combination. Among them, eukaryotic DNA is divided into chromosomal DNA and organelle DNA. The former is located in the nucleus, accounting for about 95%, and is a double-stranded linear molecule; the latter is present in organelles such as mitochondria or chloroplasts, accounting for about 5%. In addition, there are double-stranded circular plasmid DNA in prokaryotes; in acellular virus particles, DNA exists in many forms, including double-stranded, single-stranded, double-stranded, and single-stranded. The total length of DNA molecules varies greatly among different organisms, and usually increases with the evolution of organisms. For example, the composition of human DNA is about 3.0×109bp&40; blood pressure #41; the ratio of simian virus 40 to simian virus 40 is 5243bp; SV40 and #41; it is about 5.7 meters long, 10.5 times that of DNA, RNA is smaller than DNA many. Due to the diversity of RNA functions, the types, sizes and structures of RNA are more diverse than those of DNA. The difference between DNA and RNA determines the best separation and purification conditions
1. Selection of materials and methods
40;1) Selection of materials and methods
Common clinical specimens include blood, urine, saliva, tissues and cultured cells; there are many methods for nucleic acid separation and purification. How to collect and prepare materials correctly is the primary issue. The first thing to be clear is that the separation and purification of nucleic acids is not the ultimate goal. Different experimental studies and applications have different requirements for the yield, integrity, purity and concentration of nucleic acids. The time and cost of nucleic acid separation and purification are often considered; on the premise of not affecting the quality of nucleic acid, safe and non-toxic reagents and solutions are selected. In recent years, the development of kits and the use of automated instruments have made batch preparation of nucleic acid samples possible, greatly improving the efficiency of separation and purification.
(2) Selection principle
There are many methods for nucleic acid separation and purification. Different separation schemes should be adopted according to the nature and initial dosage of the specific biological material, the nature and use of the nucleic acid to be separated. Regardless of the method used, the general principles are: first, ensure the integrity of the primary structure of nucleic acid, because a complete primary structure is the most basic requirement for studying the structure and function of nucleic acid; the second is to try to eliminate the contamination of other molecules. In order to ensure the purity of nucleic acid samples, this is the main content discussed in this chapter 40; 3) Maintain the integrity of nucleic acid
In order to ensure the integrity of nucleic acid, various harmful factors should be avoided as much as possible during surgery. There are many factors that affect the integrity of nucleic acids, including physical, chemical, and biological factors, some of which can be avoided. For example, too much acid or too much base will destroy the phosphodiester bond in the nucleic acid chain. In the nucleic acid extraction process, it can be avoided by using a suitable buffer and always controlling the pH value between 4-10; in addition, if heating at high temperature, in addition to the high temperature itself destroying the chemical bonds in the nucleic acid molecule, Liquid shearing may be caused by boiling, so nucleic acid extraction is usually carried out at 0~4°C. ℃.
Secondly, for unavoidable harmful factors, various measures should be taken to minimize the damage of various harmful factors to nucleic acid. Simplify the separation and purification steps as much as possible, shorten the extraction time, and reduce the damage to the integrity of nucleic acid by various harmful factors; the activation of DNase or DNAase requires divalent metal ions such as mg2 and ca2. If EDTA and citrate are used, operate at low temperature , The activity of DNase will be significantly inhibited. The extensiveness and inactive characteristics of RNase (RNase or RNase) determine that biodegradation is the main hazard of RNA extraction. Nucleic acid extraction magnetic beads expert http:
2. Technical route design; 1) release of nucleic acid
Under normal circumstances, both DNA and RNA are located in the cell, so the first step of nucleic acid
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July 08, 2021
