Design and principles of nucleic acid isolation and purification
Nucleic acids in cells include both DNA and RNA, and both bind to proteins to form nucleoproteins. DNA and protein bind to deoxyribonucleoprotein (DNP), and RNA and protein bind to ribonucleoprotein (RNP). The DNA of eukaryotes 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%, and is a double-stranded circular molecule. In addition, there are double-stranded circular plasmid DNA in prokaryotes; in non-cell type virus particles, DNA exists in various forms, including double-stranded, single-stranded, double-stranded Shape and single-chain line. The total length of DNA molecules varies widely from species to organism and generally increases with the evolution of the organism. For example, human DNA consists of approximately 3.0×109 base pairs (bp), which is about 5.7×105 times longer than the 5243 bp simian virus 40 (SV40). Relatively speaking, RNA molecules are much smaller than DNA molecules. Since the function of RNA is diverse, the type, size and structure of RNA are more diverse than DNA. The difference in the nature of DNA and RNA determines that the optimal separation and purification conditions are different. Choice of materials and methods (1) Choice of materials and methods Common clinical specimens include blood, urine, saliva, tissue and cultured cells; there are many methods for nucleic acid separation and purification. How to properly collect and prepare materials is a primary problem. First of all, we should clarify that the separation and purification of nucleic acids is not the ultimate goal. Different experimental studies and applications may have different requirements on the yield, integrity, purity and concentration of nucleic acids; as for the time and cost of isolating and purifying nucleic acids. It is often necessary to consider; safe and non-toxic reagents and protocols should be selected without affecting the quality of the nucleic acid. In recent years, the development of kits and the use of automated instruments have enabled the preparation of nucleic acid samples in batches, greatly improving the efficiency of separation and purification. (2) The principle of choice There are many methods for nucleic acid isolation and purification, and different protocols should be adopted depending on the nature of the specific biological material and the starting amount, the nature and use of the nucleic acid to be separated. Regardless of the method used, the general principle should be followed: First, to ensure the integrity of the primary structure of the nucleic acid, because the complete primary structure is the most basic requirement for the study of nucleic acid structure and function; the second is to eliminate the pollution of other molecules as much as possible. To ensure the purity of nucleic acid samples, this is the main content discussed in this chapter. (3) Maintenance of nucleic acid integrity In order to ensure the integrity of the nucleic acid, in the course of operation, the destruction of nucleic acids by various harmful factors should be avoided as much as possible. 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, if the acid or the base is too alkaline, it has a destructive effect on the phosphodiester bond in the nucleic acid chain. In the extraction process of the nucleic acid, a suitable buffer is used, and the pH is always controlled between 4 and 10, which can be well avoided. Hazard; In addition, if heated at a high temperature, in addition to the high temperature itself damage to the chemical bonds in the nucleic acid molecule, liquid shearing may be caused by boiling, so nucleic acid extraction is often carried out at 0 to 4 °C. Shanghai Chuangsai Technology provides deoxyribonuclease (bovine pancreas), DNase I (Deoxyribonuelease I)|9003-98-9, product number: B11000133-15ku, the price is 314.3 yuan. Secondly, for the unavoidable harmful factors, various measures should be taken to minimize the damage of 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 alleviated; the activation of DNase or DNAase requires divalent metal ions such as Mg2+ and Ca2+, if EDTA or citric acid is used. The salt is operated under low temperature conditions to substantially inhibit the activity of DNase. The widespread and inactive characteristics of RNase (RNase or RNAase) determine that biodegradation is a major hazard in RNA extraction. Shanghai Chuangsai Technology provides human recombinant human RNase A / Ribonuclease A / RNASE1 protein (His tag) | Ribonuclease, RNase A family, 1 (pancreatic) Protein, product number: C86-134-68-50ug, price 2210 yuan. 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