Section 1 Design and Principles of Nucleic Acid Isolation and Purification
Nucleic acids in cells include both DNA and RNA molecules, all of which bind to proteins to form nucleoproteins. DNA and protein bind to deoxyribonucleoprotein (DNP), and RNA and protein bind to ribonucleoprotein (RNP). Among them, 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 between different organisms and generally increases with the evolution of the organism. For example, human DNA consists of approximately 3.0 × 10 9 base pairs (bp), which is about 5.7 × 10 5 times longer than the 5 243 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 the optimal separation and purification conditions for the two.
First, the 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, we should clarify that the isolation 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. The time and cost required to separate and purify nucleic acids are also 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 schemes 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 exclude other molecules from contamination 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. Such as peracid or over-base, it has a destructive effect on the phosphodiester bond in the nucleic acid chain. In the extraction process of nucleic acid, using a suitable buffer and always controlling the pH between 4 and 10, it can be well avoided. Hazard; In addition, if heated at a high temperature, in addition to the destruction of the chemical bonds in the nucleic acid molecules by the high temperature itself, liquid shearing may be caused by boiling, so nucleic acid extraction is often carried out at 0 to 4 °C.
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 Mg 2+ and Ca 2+ , if used EDTA, citrate and operation under low temperature conditions can 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. Nucleic Acid Extraction Magnetic Beads Expert http://
Second, the design of the technical route
(1) Release of nucleic acids
Under normal circumstances, both DNA and RNA are located in the cell, so the first step in nucleic acid isolation and purification is to break the cell and release the nucleic acid. There are many methods for cell disruption, including mechanical and non-mechanical methods. The mechanical method can be further divided into a liquid shearing method and a solid shearing method. The main hazard of mechanical shearing is high molecular weight linear DNA molecules, so this method is not suitable for the separation and purification of chromosomal DNA. Non-mechanical methods can be divided into drying method and lysis method. At present, most lysis methods are used. The lysis method using suitable chemical reagents and enzymatic lysing cells has wide application efficiency because of high cleavage efficiency, mild method, high yield and good maintenance of nucleic acid integrity.
(II) Isolation and purification of nucleic acids
Cell lysates are complex mixtures of nucleic acid-containing molecules that themselves may still bind to proteins. Under the premise of ensuring the integrity of nucleic acid molecules, it is not easy to separate a certain amount of nucleic acid molecules that meet the purity requirements. This requires us to fully understand the properties of nucleic acid molecules. An efficient scheme is designed to separate nucleic acids from other substances in one or more properties. This difference is multifaceted, including differences in cell localization and tissue distribution, differences in physicochemical properties, and their unique biological properties. The contaminants that should be removed mainly include three parts: non-nucleic acid macromolecular contaminants, undesired nucleic acid molecules, and solutions and reagents that are added to the subsequent experiments and applications during the separation and purification of nucleic acids. Non-nucleic acid macromolecular contaminants mainly include proteins, polysaccharides and lipids; non-required nucleic acid molecules refer to RNA as a contaminant when preparing DNA, and when DNA is prepared as a contaminant, when preparing a specific nucleic acid molecule Other nucleic acid molecules are contaminants; as for the organic solvent and some metal ions added during the separation and purification of nucleic acids, they often need to be well removed due to their influence on subsequent experiments. Nucleic Acid Extraction Magnetic Beads Expert http://
(3) Relationship between nucleic acid quality and extraction steps
In general, the more separation and purification steps, the higher the purity of the nucleic acid, but the yield will gradually decrease, and the integrity will be more difficult to ensure. On the contrary, by separating the experimental scheme with few purification steps, we can obtain more nucleic acid molecules with better integrity, but the purity is not necessarily high. This requires selection in conjunction with the use of the nucleic acid.
(4) Concentration, precipitation and washing of nucleic acids
With the gradual addition of nucleic acid extraction reagents and the inevitable loss of nucleic acid molecules during the removal of contaminants, the concentration of nucleic acids in the sample will gradually decrease, and will affect the subsequent experimental operations or meet the needs of subsequent research and application. The nucleic acid is concentrated. Precipitation is the most commonly used method for nucleic acid concentration. It has the advantage that after the nucleic acid is precipitated, the lysis buffer can be easily changed and the nucleic acid solution can be adjusted to the desired concentration. In addition, the nucleic acid precipitate can also remove some impurities and certain salt ions. Purification. After adding a certain concentration of the salt, the nucleic acid is precipitated with an organic solvent. Among the commonly used salts are sodium acetate, potassium acetate, ammonium acetate, sodium chloride, potassium chloride and magnesium chloride. Commonly used organic solvents are ethanol, isopropanol and polyethylene glycol. Nucleic acid precipitation often contains a small amount of coprecipitated salt, which needs to be washed and removed with 70% to 75% ethanol. For low-density and bulky nucleic acid samples, they can be concentrated using solid polyethylene glycol or butanol before precipitation in organic solvents.
Third, identification, preservation and application
(1) Identification of nucleic acids
1. Concentration Identification Quantitative identification of nucleic acid concentrations can be performed by UV spectrophotometry and fluorometry.
(1) Ultraviolet spectrophotometry: Ultraviolet spectrophotometry is based on the fact that the bases in the molecular components of the nucleic acid have certain ultraviolet absorption characteristics, and the maximum absorption wavelength is between 250 nm and 270 nm. When these bases form nucleotides with pentose or phosphoric acid, their maximum absorption wavelengths are unchanged. After the nucleic acid is composed of nucleotides, its maximum absorption wavelength is 260 nm, which lays a foundation for determining the concentration of nucleic acid in the solution. Under ultraviolet light having a wavelength of 260 nm, the optical density of one OD value corresponds approximately to 50 μg/ml of double-stranded DNA, 38 μg/ml of single-stranded DNA or single-stranded RNA, and 33 μg/ml of single-stranded oligonucleotide. If the concentration of a single-stranded oligonucleotide molecule of a known sequence is to be accurately quantified, it must be calculated in accordance with Lambert-Beer's law in combination with its actual molecular weight and molar absorptivity. If the DNA sample contains salt, the reading of A 260 will be too high. A 310 should be determined to subtract the background, and the difference between A 260 and A 310 should be used as the basis for quantitative calculation. Ultraviolet spectrophotometry is only used to determine nucleic acid solutions at concentrations greater than 0.25 [mu]g/ml.
(2) Fluorescence spectrophotometry: Fluorescence spectrophotometry, after the fluorescent dye ethidium bromide (EB) is inserted into the base plane, the nucleic acid which is not fluorescent itself emits orange-red fluorescence under ultraviolet excitation, and the fluorescence intensity The integral is proportional to the nucleic acid content. The sensitivity of the method can reach 1 ng to 5 ng, which is suitable for quantitative analysis of low concentration nucleic acid solution. In addition, SYBR Gold is a new ultra-sensitive fluorescent dye that can detect less than 20pg of double-stranded DNA from an agarose gel.
2. Purity Identification Ultraviolet spectrophotometry or fluorescence spectrometry can be used for the purity identification of nucleic acids.
(1) Ultraviolet spectrophotometry: Ultraviolet spectrophotometry mainly determines the presence or absence of protein contamination by the ratio of A 260 to A 280 . In TE buffer, the pure DNA had an A 260 /A 280 ratio of 1.8 and the pure RNA had an A 260 /A 280 ratio of 2.0. Both the increase and decrease in the ratio indicate impure. Both the protein and the phenol added in the nucleic acid extraction reduce the ratio. The high absorption peak of the protein at 280 nm and the phenol at 270 nm can identify whether it is mainly protein contamination or phenol contamination. The contamination of RNA can cause the ratio of DNA products to be higher than 1.8, so the DNA solution with a ratio of 1.8 is not necessarily a pure DNA solution, and may be contaminated with protein, phenol and RNA, and needs to be identified by other methods. The ratio of A 260 /A 280 is a good indicator of the degree of protein contamination, and 2.0 is a hallmark of high quality RNA. However, it should be noted that due to the different secondary structure of RNA, the reading may have some fluctuations, generally between 1.8 and 2.1 is acceptable. In addition, the pH of the solution used to identify RNA purity affects the A 260 /A 280 reading. For example, the A 260 /A 280 ratio of RNA in aqueous solution is 0.2 to 0.3 lower than its reading in Tris buffer (pH 7.5). Nucleic Acid Extraction Magnetic Beads Expert http://
(2) Fluorescence spectrophotometry: The result of nucleic acid electrophoresis traced with a fluorescent dye such as ethidium bromide can be used to determine the purity of the nucleic acid. Because DNA molecules are much larger than RNA, electrophoretic mobility is low; RNA has the largest rRNA, accounting for 80%-85%, tRNA and nuclear small RNA accounting for 15%-20%, and mRNA accounting for 1%-5%. Therefore, the total RNA can exhibit characteristic three bands after electrophoresis. In the prokaryote, there are clearly visible 23S, 16S rRNA bands and a relatively diffuse fast-migrating band composed of 5S rRNA and tRNA. In eukaryotes are 28S, 18S rRNA and a band consisting of 5S, 5.8S rRNA and tRNA (Figure 6-1). The lack of mRNA and the size of the molecules are generally invisible. By analyzing the results of nucleic acid gel electrophoresis using ethidium bromide as a tracer dye, we can identify the presence or absence of RNA interference in DNA products, and also identify the presence or absence of DNA contamination in RNA products.
3. Integrity Identification Gel electrophoresis is routinely used.
(1) Gel electrophoresis: The results of nucleic acid gel electrophoresis using ethidium bromide as a tracer dye can be used to determine the integrity of nucleic acids. Genomic DNA has a large molecular weight and is very slow to move in an electric field. If a small molecule DNA fragment is degraded, it can be significantly expressed on the electropherogram. The complete total RNA electropherogram without degradation or degradation, except for the characteristic three bands, the fluorescence intensity integral of the three bands should be a specific ratio. The nucleic acid band with large sedimentation coefficient has large molecular weight, low electrophoretic mobility and high fluorescence intensity integral; on the contrary, the molecular weight is small, the electrophoretic mobility is high, and the fluorescence intensity integral is low. Generally, 28S (or 23S) RNA has a fluorescence intensity about twice that of 18S (or 16S) RNA, otherwise it suggests degradation of RNA. If there is a colored band near the loading slot, it indicates DNA contamination. Nucleic Acid Extraction Magnetic Beads Expert http://
(2) Other methods: If necessary, RNA can be analyzed by special tests, such as small-scale first-strand cDNA synthesis reaction, radiolabeled oligo-deoxythymidine oligo (dT) as a probe Northern hybridization and Northern hybridization to mRNA of known size. In addition, with the rapid development of capillary electrophoresis and biochip technology, the means of separation, purification, identification and recovery of nucleic acids are increasingly abundant.
(2) Preservation of nucleic acids
The structure and properties of the nucleic acid are relatively stable, and it is not necessary to prepare a fresh nucleic acid sample every time, and the nucleic acid sample prepared at one time can often meet the needs of multiple experimental studies, so it is necessary to investigate the storage environment and conditions of the nucleic acid. As with isolation and purification, the storage conditions of DNA and RNA vary depending on the nature.
1. Preservation of DNA For DNA, it can be stored in TE buffer at -70 ° C for several years. Among them, the pH value of TE is 8, which can reduce the deamination reaction of DNA, and the DNA is easily denatured when the pH is lower than 7.0; EDTA acts as a chelating agent for divalent metal ions by chelation of divalent metal ions such as Mg 2+ and Ca 2+ . In order to inhibit the activity of DNase; low temperature conditions are beneficial to reduce various reactions of DNA molecules; double-stranded DNA is highly inert due to structural characteristics, and can be stored for a long time at 4 ° C; A small amount of chloroform can effectively prevent bacterial and nucleic acid contamination. Nucleic Acid Extraction Magnetic Beads Expert http://
2. RNA storage RNA can be dissolved in 0.3 mol/L sodium acetate solution or double-digested water, and stored at -70 ° C to -80 ° C. If RNA is dissolved in diethyl pyrocarbonate (DEPC) or RNasein or vanadyl-ribonucleoside complex (VRC) is added to the RNA solution, Inhibition of RNase degradation of RNA and prolonged storage time. In addition, the RNA precipitate is dissolved in a 70% ethanol solution or a deionized formamide solution and can be stored at -20 ° C for a long time. Among them, the formamide solution can avoid the degradation of RNA by RNase, and the RNA is very soluble in the formamide solution, and its concentration can be as high as 4 mg/ml. It should be noted that the addition of these so-called RNase inhibitors or organic solvents is only a temporary preservation requirement and must be removed if they have an impact on subsequent experimental research and application.
Since the mechanical shear force generated by repeated freezing and thawing has a destructive effect on DNA and RNA nucleic acid samples, in practice, a small amount of nucleic acid is very necessary.
(C) the application of nucleic acids
The isolated and purified nucleic acid sample can be used for the study of the function and properties of the nucleic acid itself, and can also be widely used for its functions and properties, such as genetic engineering and protein engineering using nucleic acid molecules as the starting material. It can be said that most molecular biology techniques including polymerase chain reaction, nucleic acid hybridization and DNA recombination and expression technology are all based on nucleic acid molecules, using the base pairing principle of nucleic acid molecules and nucleic acids. Performing one or more enzyme modifications. The vast majority of molecular biology techniques are essentially the analysis of DNA and RNA. Without the isolation and purification of nucleic acids, molecular biology techniques have no basis for research and application. Nucleic Acid Extraction Magnetic Beads Expert http://
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