Nucleic acid extraction experience and principle

1. Principle of nucleic acid extraction

Briefly, nucleic acid extraction includes two major steps of sample lysis and purification. Lysis is the process of freeing the nucleic acid in the sample in the lysis system, while purification is the process of completely separating the nucleic acid from other components in the lysis system, such as proteins, salts and other impurities. Classical lysates almost all contain detergents (such as SDS, Triton X-100, NP-40, Tween 20, etc.) and salts (such as Tris, EDTA, NaCl, etc.). The role of salt, in addition to providing a suitable lysis environment (such as Tris), also includes inhibiting nucleic acid damage (such as EDTA) by nucleases in the sample during the lysis process, maintaining the stability of nucleic acid structure (such as NaCl), etc. Detergent is to denature the protein, destroy the membrane structure and untie the protein connected with the nucleic acid, so that the nucleic acid is free in the lysis system. Protease may also be added to the cleavage system; the protein is digested into small fragments by protease, which promotes the separation of nucleic acid and protein, and at the same time, facilitates subsequent purification operations and obtains purer nucleic acid. There are also lysates directly using high concentrations of protein denaturants (such as GIT, GuHCl, etc.). This method has become the mainstream of RNA extraction, but not the mainstream of genomic DNA extraction.

The most commonly used purification methods are PC extraction + alcohol precipitation and media purification. The first method is to use PC to repeatedly extract the cleavage system to remove proteins to achieve the separation of nucleic acids and proteins; and then use alcohol to precipitate nucleic acids to achieve the separation of nucleic acids and salts. The second method is to use some solid media to selectively adsorb nucleic acids under certain specific conditions, but not to adsorb proteins and salts, so as to realize the separation of nucleic acids from proteins and salts. Protein removal by high-salt precipitation is a variant of the first purification method that omits the hassle of PC manipulation. Of course, there are also extraction methods without purification, but their use is mostly limited to simple PCR. The removal of other impurities - such as polysaccharides, polyphenols, etc. - is basically achieved by adding some special reagents or adding some additional steps on the basis of these two methods.

2. Know your experimental samples

If you study an experimental sample and want to extract its nucleic acid, the following information must be collected first: the nucleic acid content, enzyme content, and special impurity content of the sample. If you don't know anything about the characteristics of the sample, when the sample is a little complicated, the experiment of extracting nucleic acid will encounter many problems. Taking blood as an example, if you do not know that the content of nucleated cells in bird blood is about a thousand times that of human blood, and the same starting amount of human blood is used to extract genomic DNA from bird blood, how can it be successful? If you fail, how do you know why? At the same time, only with an understanding of the experimental sample can the correct choice of the lysis method be made. In most cases, the best results are obtained with fresh samples. For some samples with high impurity content, if the genomic DNA is extracted from fresh samples and encounters the problem of excessive residual impurities, you can try the countermeasures of -20C storage for one day before extraction, which may have unexpected results. If the sample must be stored first for some reason, the sample should also be simplified first: it is best to store only nucleated cells in blood; the sample should be divided and stored to avoid repeated freezing and thawing. If the laboratory does not have suitable storage conditions, it is a good choice to lyse the sample first and then store it.

3. Evaluation of the cracking method

Protease-containing cleavage methods can be considered the first choice for the extraction of genomic DNA. Cleavage includes the release of membrane proteins and the release of proteins linked to genomic DNA. The role of protease is to make the protein smaller, so it has a huge promotion effect on the free protein; at the same time, the huge genomic DNA is easy to "entangle" the macromolecules, and after the protein is digested by the protease, it is not easy. Being "entangled" by genomic DNA is conducive to the removal of proteins during purification operations, so that the purity of the final genomic DNA is higher.

Another idea is that if the genomic DNA is "entangled" with the protein, there are two possibilities during the purification process: if the characteristics of the genomic DNA are dominant, it will be retained in the form of DNA during purification, resulting in the residue of the protein; If the properties of the protein predominate, it is removed in the form of protein during purification, resulting in loss of DNA. For some samples, such as muscle, even for RNA extraction, the use of a protease-containing lysis buffer (or protease digestion of the protein at some point in the procedure) is strongly recommended because the protein in these samples is very difficult to remove. This method is the basis for obtaining maximum yield and purity. Detergent lysis method without protease,

There are still advantages in cellular genomic DNA extraction, especially when yield and purity requirements are not the highest, and economy and simplicity of operation are important. Controlling the lysate/sample ratio is critical to the success of this method. This method, combined with high-salt precipitation, allows for the simplest operation, but may be less stable in purity and yield than extraction with PC. The lysis method of high concentration protein denaturant (such as GIT, GuHCl, etc.) is the first choice for RNA extraction. For the extraction of total RNA, the most important thing is to quickly lyse the cell membrane. As for the cleavage of the protein connected to the genomic DNA and the problem of "entanglement" between the genome and the protein, it will not have a great impact on the subsequent purification. consider. High concentrations of protein denaturants can rapidly destroy cell membranes, thereby rapidly inhibiting intracellular RNases, thereby ensuring RNA integrity. With the exception of very few samples for which this method is not applicable - mainly plants, the extraction of RNA from most samples can be based on high concentrations of protein denaturants. This method can also be used for genomic DNA extraction and is very fast and simple, but the purity is not very high.

The CTAB-containing lysate has almost become the preferred lysis method for the extraction of genomic DNA from polysaccharide-rich samples such as bacteria and plants. The success of this method is related to two factors: one is the quality of the CTAB, and the other is the thoroughness of the washing. The quality of CTAB has a great influence on the cracking efficiency, and it seems unclear why, because even CTAB of the same purity produced by the same company, the effect may vary greatly depending on the batch number. It is more difficult to remove CTAB by washing than other salts, and at the same time, a small amount of CTAB residue will have a huge impact on the enzyme activity, so the thorough washing is also the key to the success of this method. For the pyrolysis temperature, use 65C; but if you find that the degradation is severe or the yield is too low, you can try the relatively low temperature region of 37C – 45C. SDS alkaline lysis method is the preferred lysis method for plasmid extraction, which has the characteristics of rapidity, high yield and almost no genomic DNA contamination. Controlling the ratio of lysate/cell and mild operation are the keys to the success of this method.

The protein precipitation efficiency is better at 4C, so the quality can be improved by adding solution III and standing at 4C for a period of time and centrifuging at 4C to remove the protein. This method does not necessarily require the use of PC purification, but in combination with PC purification, very high-purity plasmids can be obtained. Removal of RNA can be accomplished by adding RNase A (100ug/ml) to solution I or RNase A (25ug/ml) to the final lysate. The general feeling is that with RNase A in solution I, there is less residual RNA. However, classical precipitation is almost impossible to completely remove RNA residues. Additionally, this method can be problematic for large plasmids (above 50 kb). The simple cleavage method of PCR template is also a widely used method.

The feature of this method is that no purification is required, and the lysate can be directly taken for PCR after the sample is lysed, which is very fast. Because of the lack of purification, the proportion of false negatives (that is, positives that are not amplified) is relatively high. The simplest lysis solution in this method is water. The more complex ones will contain some things that will not inhibit the subsequent PCR reaction, but can improve the lysis efficiency, and may even partially eliminate the impurities in the sample that inhibit the PCR reaction, such as Triton X-100 , formamide, etc. A little more complex will contain media such as Chelex 100 that can absorb some of the impurities.

The operation is also very simple, and the temperature change is often used to achieve sample cracking, such as boiling, or multiple cycles of high temperature and low temperature. This method is most suitable for finding positive samples from a large number of samples, but it is not suitable for determining whether a sample is positive or negative. Reducing the amount of sample used can improve the positive rate, because the reduction of the sample amount also means that the amount of PCR inhibitor is reduced. After choosing a suitable lysate, the next step is to control the ratio of sample to lysate.

This issue is very important, but it has not received enough attention. Serious references should provide a simple ratio, eg 1ml of lysate can be used for T mg tissue or C cells; my suggestion is that your sample volume is definitely smaller than the data provides. There is no specific statement on the size of the starting sample. If the amount of samples is not limited, it is reasonable to use the amount of nucleic acid that can be extracted to meet the requirements of several successful experiments as the basis for determining the initial amount of samples.

Do not use 100 mg of sample just because 1 ml of lysis buffer can extract 100 mg of sample. The amount of the lysate is apparently not related to the extraction results (purity and yield), however, in practice, it has a relatively large impact on the results. The principle of the amount of lysis solution is to ensure that the sample can be completely lysed, and at the same time, the concentration of nucleic acid in the lysis system is moderate. If the concentration is too low, the precipitation efficiency will be low and the yield will be affected; if the concentration is too high, the process of removing impurities will be complicated and incomplete, resulting in a decrease in purity. Also, keep in mind that the amount of lysate used is based on the protein content of the sample, not the nucleic acid content.

4. Purification method

Evaluating PC extraction/alcohol precipitation methods is a timeless method. Stable, reliable, economical and convenient. PC extraction can completely remove proteins, and alcohol precipitation can remove salts. For general clean samples (impurities are proteins), this method can completely obtain high-quality nucleic acids. Although part of the nucleic acid will be lost in each PC extraction (because it is impossible to pipet all the aqueous phase), and the alcohol precipitation of low concentration nucleic acid will be inefficient, but these problems can be solved or reduced by adjustment of the operation. The biggest problem with this method is that it is not suitable for large-scale extraction. PC extraction is a very effective means of removing proteins. Phenol can denature the protein, and the denatured protein is precipitated from the aqueous phase, in the phenol or between the phenol/water phase.

The key to PC extraction is to mix thoroughly, and secondly, to use a sufficient amount. Mix thoroughly to ensure adequate contact between phenol and protein to fully denature the protein. Many people are always concerned that the intensity of mixing will damage nucleic acids, especially genomic DNA, but it is not necessary to be so careful. Vigorous mixing operation will partially disrupt the genomic DNA of macromolecules, but the disruption will not be so strong that the DNA becomes small fragments within 10kb. After shaking and mixing vigorously by hand, most of the genomic DNA fragments will be larger than 20kb. This size, except for some special requirements, is completely suitable for PCR and enzyme digestion.

If the required fragments are very large, such as for library construction, vigorous mixing methods cannot be used, but gentle back-and-forth inversion – the key here is that the proportion of lysate is large enough that the system is not too viscous . The amount should be sufficient, because the removal of protein by phenol has a certain degree of saturation. If the saturation is exceeded, the protein in the cleavage system will not be removed once, and must be completely removed by multiple extractions. In addition, the disadvantage of a too viscous system is that the protein is difficult to remove completely, and the genomic DNA will be more fragmented, so pay attention to the ratio of lysate to sample. A 4C centrifugation procedure facilitates more complete protein removal.

Another use of PC extraction is that the use of acidic phenols can partially remove the characteristics of DNA and obtain RNA with very little DNA residue during RNA extraction. However, it should be reminded that some plant samples cannot be extracted with PC before removing certain impurities, otherwise the nucleic acid will be degraded. High salt precipitation protein/alcohol precipitation method is also a very good method. Compared with the PC extraction method, this method overcomes almost all the disadvantages of PC extraction except that the stability of the purity may be lower.

The concomitant benefit of faster and easier protein removal is that it can be used for large scale extractions, the disadvantage is that the purity (protein residue) is not stable enough. Protein precipitation efficiency is better at 4C. The medium purification method is an increasingly important method. Its biggest feature is that it is very suitable for large-scale nucleic acid extraction, and because it is less affected by human operating factors, the stability of the purity is very high (although the purity is not necessarily higher than the PC purification method). Its Achilles heel is sample excess.

Media can be divided into two categories, one is column type, that is, the medium is pre-packed in a column with a pass below; the other is granular (such as Glassmilk, magnetic beads, etc.). The purification operation of the granular medium is not much different from the classical alcohol precipitation. It is through several additions and pouring processes. After drying, the purified nucleic acid can be obtained by dissolving. Although the operation of column purification also has the process of adding liquid and pouring liquid, because the added liquid will enter another centrifuge tube after centrifugation, which is completely separated from the column containing nucleic acid, so the washing is more thorough and the operation is more labor-saving ( Don't worry about pouring out the nucleic acid, or the residue of the liquid). However, the cost of the media purification method is the highest.

5. Precipitation of alcohol

The purpose of alcohol precipitation is to precipitate nucleic acids from the lysis system, thereby separating nucleic acids from other impurities, mainly salts. In practice, many impurities are also precipitated by alcohol together with nucleic acid, especially when the concentration of other impurities is relatively high. Alcohol precipitation is not very specific, and any organic macromolecules and some salts may be simultaneously precipitated when the concentration reaches a certain level. As far as nucleic acid is concerned, standard alcohol precipitation requires a certain amount of salt and a certain proportion of alcohol, but this by no means means that these salts are indispensable or that the proportion of alcohol cannot be changed.

In practice, it is not difficult to find that when the concentration of nucleic acid in the lysis system reaches a certain level, even if the system does not contain the salt recommended in the textbook, the nucleic acid can be precipitated by using alcohol alone; Nucleic acids can be precipitated (of course, yields may be reduced). The point of knowing this is: Don't be superstitious about the uniqueness of standard methods; instead, when you run into problems with standard methods—primarily purity issues—they can be improved by adjusting the precipitation conditions.

The most valuable reference is a precipitation solution provided by TRIzol: replacing pure isopropanol with half isopropanol and half high-salt solution, which can greatly reduce polysaccharide residues. Another problem is that it must be firmly believed that the alcohol precipitation process of nucleic acid is also the precipitation process of other impurities; although adjusting the conditions of alcohol precipitation will reduce the yield of nucleic acid, it can greatly improve the purity. If the starting sample for nucleic acid extraction is relatively "dirty", in principle, cryoprecipitation should not be used. Low temperature precipitation can improve the precipitation efficiency: when the nucleic acid concentration is very low, the effect is obvious; when the nucleic acid concentration is relatively high, the effect is not obvious, but it will lead to a great increase in impurities.

I haven't seen a big impact on quality whether alcohol precipitation uses isopropanol or ethanol, although many people "find" that ethanol precipitated nucleic acids to be more pure. The nucleic acid precipitated by isopropanol is relatively compact, the wall is tight, and the color is not very white; the nucleic acid precipitated by ethanol is relatively fluffy, easy to move from the wall, and the color is relatively white. This is a phenomenon, suggesting the conclusion: nucleic acids precipitated by isopropanol are not easily discarded, but are more difficult to wash.

Combining the two aspects of loss and washing, it is recommended that a small amount of nucleic acid be precipitated with isopropanol (a small amount, washing is not a problem, and no loss is the first), a large amount of nucleic acid is precipitated with ethanol (a large amount, loss is not a problem, washing is convenient for First). As for the statement that isopropanol precipitation is easier to precipitate salt, I have never encountered it (I hardly use low temperature precipitation, is it easier for low temperature precipitation to cause salt precipitation?); I think the reason for this phenomenon is that the washing process is not good. thorough. Of course, don't forget that the biggest advantage of isopropanol precipitation is its small size, which allows most of the small extraction operations to be completed in a 1.5ml centrifuge tube.

However, because the precipitate is very compact, the central part of the precipitate is not easy to be washed. Therefore, the key to washing nucleic acid precipitated by isopropanol is: the precipitate must be suspended, and it must be placed for a period of time to make the precipitate finally fluffy. white. If you wash it again, the quality will never be a problem. PEG, LiCl, CTAB can all be used for nucleic acid precipitation. Although they are far from the high frequency of use of alcohol precipitation, they have their own characteristics. LiCl precipitates RNA to remove DNA, and CTAB precipitates nucleic acids from polysaccharide-containing lysis systems. PEG is a convenient means of precipitating viral particles.

6. Washing

Washing must first suspend the precipitate; the second is to have a certain amount of time, especially when the nucleic acid precipitate is relatively large (to make the nucleic acid precipitate finally fluffy); the third is a small amount of time; the fourth is to remove the supernatant thoroughly . The operation in the textbook is basically "discard the supernatant and put it upside down on the absorbent paper for a while". This description itself is not problematic and very convenient, but because he is from abroad, he naturally has the problem of "acclimatization". If the centrifuge tube is siliconized, because the liquid hardly hangs on the wall, the supernatant can be removed very thoroughly; for a good tube, even if it is not siliconized, the residual amount is very small, and there is no problem; for a poor tube, the liquid The wall hanging is very impressive, and the residual amount is so large that it will affect subsequent experiments. The only operation that is not affected by the quality of the tube is to pour off the liquid and then briefly centrifuge to remove the residual liquid with a pipette.

It must be kept in mind that the residual liquid contains impurities from the previous operation, and the residual amount is related to the degree of mixing and the size of the nucleic acid precipitate. Ethanol is removed during volatilization, and impurities will not be removed by volatilization. In addition, the size of the nucleic acid pellet and the lysis capacity of the lysate also determine the intensity of washing. The larger the precipitation, the stronger the lysing ability of the lysing solution, and the more thorough the washing is: the storage time is relatively longer, and the number of washings should also be increased. Most importantly, wash with 75% ethanol at room temperature.

7. Nucleic acid dissolution and preservation

The purified nucleic acid is finally dissolved in water or low-concentration buffer; the RNA is mainly dissolved in water, and the DNA is dissolved in weakly alkaline Tris or TE. The classical DNA solubilization method advocates the use of TE solubilization. It is believed that EDTA can reduce the risk of DNA being degraded by DNase that may remain; if the operation process is properly controlled, the residue of DNase is almost negligible, and Tris or water (pH) can be used directly. Approach 7) Solubilize DNA. Basically, the stability of nucleic acids in storage is inversely proportional to temperature and proportional to concentration. Although some experimenters have found that DNA stored at -20C is more stable than -70C, I would rather consider this an exception.

If the temperature is suitable, the nucleic acid will degrade or disappear during storage. The first reason is the enzymatic hydrolysis caused by the residual enzyme, and the second reason is the hydrolysis caused by the inappropriate pH value of the nucleic acid solution (RNA is more stable in weak acidity, while DNA is more stable in weak acid). Weak alkaline is more suitable). Another thing that is not taken seriously is the impact of EP tubes on nucleic acids. First of all, it must be firmly believed that nucleic acid will definitely react with the contact surface of the container in which it is placed to reach a certain equilibrium. The material of the EP tube may firstly adsorb nucleic acid, and secondly, it can induce some changes in the structure of nucleic acid, such as denaturation.

When the nucleic acid concentration is relatively high, this phenomenon may not be observed; when the nucleic acid concentration is very low, it is more obvious. Adding Geletin, Glycogen, and BSA to low concentrations of nucleic acid can stabilize nucleic acid. Although it has been proved by experiments, many experimenters do not take this suggestion seriously. There are far more materials that make EP pipes now than in the past. These newly emerging materials may be much better than the original pure PP materials in terms of physical characteristics such as strength and transparency, but their chemical characteristics, especially their possible effects on nucleic acid stability, are far from thoroughly studied. Just as the current plasmids can be modified to meet certain requirements, the negative product may be that the extracted plasmids have more and more configurations in electrophoresis: in addition to the original three band types,

8. Detection of nucleic acid quality

Using the extracted nucleic acid directly for subsequent experiments is the only reliable detection method; other detection methods are relative and not very reliable. At present, the methods used to detect the quality of nucleic acid before formal experiments are electrophoresis and ultraviolet spectrophotometer. Electrophoresis mainly detects the integrity and size of nucleic acid. As long as the nucleic acid is not too small or too large (beyond the range of electrophoretic separation), this method is still very reliable; electrophoresis can also be used to estimate the concentration of nucleic acid, but its accuracy is similar to that of nucleic acid. Experience is relevant; in addition, electrophoresis may also provide information on contamination by certain impurities, but is also empirical.

UV spectrophotometers measure purity and nucleic acid content, however, because UV spectrophotometers cannot be guaranteed to be very accurate, and the sensitivity of the instrument is very high, the results provided are not very reliable. Generally speaking, a more reasonable judgment can be made by combining the results of UV and electrophoresis detection at the same time. But since both methods have flaws, it shouldn't be a fuss if bad results can be used in follow-up experiments and good results can't be used in follow-up experiments. About the detection of UV spectrophotometer.

First, don't use instruments that don't have wavelength selection; using instruments that have wavelengths fixed is probably the beginning of your nightmare. (No information is terrible, and even more terrible is to take wrong information seriously.) Second, be sure to adjust the instrument frequently; the adjustment can use very pure self-provided nucleic acid, or you can use phenol (the latter is my current every time The method used in both methods is very simple; see my previous post for details.) Finally, remember that the readings A230 : A260 : A280 = 1 : 2 (DNA is 1.8) : 1 are theoretical data, which will be calculated in actual measurement. There are some differences; but if the difference is too large, there is a problem.

There are two debatable points: if A260/A230 > 2 it is pure, and if A260/A280 > 2.3 it is nucleic acid degradation. If A260/A230 is much larger than 2.0, there must be impurities remaining (I don't know what the impurities are). If the nucleic acid is measured immediately after extraction, A260/A280 > 2.3 will never indicate nucleic acid degradation, because the nucleic acid fragments that can lead to A260/A280 > 2.3 are very small, and it is impossible to precipitate them by conventional precipitation; The more likely reason is that the A260/A280 provided by your instrument is actually the value of A262/A282 or even A264/A284.

The absorbance of pure nucleic acid is the bottom at A230, the peak at A260, and the half-hill at A280. According to the characteristics that the slope of the curve is the smallest at the bottom and the top, and the slope is the largest at the half-hill slope, it is not difficult to draw the following conclusion: the readings of A230 and A260 are less affected by the accuracy of the instrument, while the A280 is more affected by the accuracy of the instrument. It is recommended to first detect by electrophoresis, and then detect by UV spectrophotometer. After electrophoresis, you can see which samples cannot be used at all (such as too much degradation), as well as the approximate concentration of nucleic acid, which provides a reference for the detection of UV spectrophotometer (degradation is unnecessary to measure, the how much to take, etc.).