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1. Introduction to nucleic acid extraction and purification
Nucleic acid extraction is a basic part of molecular biology, because high-quality nucleic acid is essential for molecular biology applications.
We will summarize some of the currently used nucleic acid extraction techniques and tips on choosing the correct extraction method for the sample type; we will also mention the evaluation of nucleic acid concentration and quality, as well as common problems in the nucleic acid extraction process.
a. Why is nucleic acid extraction needed?
Nucleic acid extraction provides answers to a large number of extensive research and applications (for example: cloning, qRT-PCR and next-generation sequencing technology in the field of whole genome and transcriptome), and the obtained nucleic acid can be used in a variety of ways.
The exact research purpose determines the type of nucleic acid to be extracted; the application of nucleic acid often affects the choice of extraction method. (For example: standard End-point PCR reaction does not require the same DNA quality as a whole-genome sequencing experiment)
In order to determine the research method, it is necessary to have a clear understanding of the downstream applications of nucleic acids and any potential limitations related to the type of sample. (For example, the collection of clinical samples is often limited, and nucleic acid extraction presents challenges.)
Although the method of cell lysis is different depending on the sample type, the core principle of the overall nucleic acid extraction remains the same: cell or tissue samples are lysed to remove non-nucleic acid contaminants (for example, proteins, etc.).
Reasons for extracting nucleic acid:
①To analyze gene expression in basic research and disease research;
②Track the response to drug treatment (for example, monitor virus titer during and after antiviral treatment).
③Identify new species and gain insight into the evolutionary process (for example, Ancient DNA analysis).
④Monitor and classify pathogens that cause outbreaks of infectious diseases in humans, animals and plants.
⑤ Monitoring food and water safety through microbiological testing and quantitative monitoring.
⑥Diagnosing diseases (such as genetic diseases, cancer, immunological defects).
b. Method of cell lysis
Cell lysis largely depends on the sample type, and stronger tissues, such as plants, require more force to process than mammals.
Cell lysis methods are divided into three categories: mechanical lysis, enzymatic lysis and chemical lysis.
The basic principles of the three analysis methods and the advantages and disadvantages of each method are as follows:
Method 1: Mechanical cracking
Mechanical lysis: The cell membrane is destroyed by external force.
High efficiency and fast speed;
Fast lysis shortens the time from sample collection to nucleic acid isolation, which may be crucial in gene expression analysis experiments;
Ideal for samples that are difficult to dissolve (for example, plant material, filamentous fungi and yeast).
Depending on the method used, processing of multiple samples is time-consuming (for example, manual grinding processing);
Processing a large number of samples is time-consuming and may increase the risk of sample degradation;
Mechanical processing will generate heat in the sample, leading to protein aggregation and nucleic acid degradation; l requires some special tools or equipment.
Method 2: Enzymatic lysis
Add an enzyme to digest proteins or cell structures, such as yeast cell walls and filamentous fungi.
The ideal method in the laboratory, without mechanical cracking equipment in the process;
Selectively remove the cell wall, leaving the cell part to use another lysis method (usually chemical lysis), the method is flexible, and some damage caused by mechanical lysis is avoided.
Time-consuming (enzyme digestion may take 1 hour) and expensive;
Because enzyme-induced cell changes may affect gene expression, it is not suitable for gene expression analysis.
Method 3: Chemical lysis
In the chemical decomposition process, the cells are washed with a detergent that can break down lipid membranes, thereby releasing cellular components. In addition to detergents, chemical lysis buffers usually contain chaotropic salts, such as guanidine hydrochloride or urea, which help degrade the stability of newly exposed nucleic acid proteins (such as nucleases) and bind the nucleic acids to the silica matrix. The exact composition of the chemical lysis buffer depends on the application direction and sample type.
The price is cheap, the operation is simple, and the speed is fast; no special equipment is required.
Detergents that destroy cell membranes usually also dissolve other cell membranes, thereby releasing their components. This method is not suitable for the extraction of organelle-specific nucleic acids;
Although this technology is effective against E. coli, it is not effective against Gram-positive bacteria, plant cells or fungal cells because of the hard cell wall that prevents detergent from entering the cell membrane;
Adding detergent and chaotropic salt to E. coli samples at the same time may affect the distinguishing ability of plasmid DNA and genomic DNA. However, steps can be taken to distinguish these types, which will be discussed later.
Chemical substances can be dangerous to researchers.
c. Understanding of the principles of nucleic acid purification
After the cell is lysed, the nucleic acid is selectively released from the surrounding cell components and concentrated in an aqueous solution or a suitable buffer solution for subsequent use. The steps after cell understanding are collectively referred to as purification. Since the chemical properties of the isolated nucleic acid remain unchanged, regardless of the sample type as follows, the purification strategy described below is generally applicable to all sample types.
① Spin Columns
This method is usually closely related to the kit. Spin column extraction and purification can quickly obtain high-quality DNA, plasmids, RNA and PCR products without the need for fresh reagent preparation. The spin column contains a solid matrix of silica gel or proprietary resin for selective binding of nucleic acids.
Typical spin column extraction operation steps:
A. Keep the lysed sample on the spin column:
The lysed sample is collected in a binding buffer containing chaotropic salts and placed on a spin column. The binding buffer allows the nucleic acid to be separated from the aqueous solution and bound to the column matrix.
B. Binding of nucleic acid:
Centrifugation brings the entire sample into contact with the column matrix. Nucleic acids in the sample selectively bind to the column, while other cellular components pass through (this is commonly referred to as flow through) the column matrix.
After binding, the column is cleaned to remove any residual contaminants, such as proteins or residual salts, that may seriously affect downstream applications. The number of washes depends on the kit. Some wash buffers contain chaotropic salts to remove proteins, and some buffers contain high concentrations of ethanol to remove salts. Most kits almost entirely include a buffer composed of ethanol as a washing step to ensure complete desalination.
D. Removal of residual ethanol from the spin column:
After washing, sometimes a short period of empty centrifugation is performed to remove residual ethanol.
To elute nucleic acids from the matrix, add a small amount of water or elution buffer*, and let the column stand for a few minutes. Since the previous step, the chaotropic salt has been removed, and the elution buffer can rehydrate the nucleic acid and separate the nucleic acid from the column matrix. One centrifugation can transfer the aqueous solution containing the nucleic acid sample to a new centrifuge tube.
*Nucleic acids are usually stored in a buffer containing Tris and EDTA (TE buffer). The presence of EDTA helps to inhibit nuclease activity. Although TE buffer is effective in inhibiting nucleases, the content of EDTA in TE is usually several orders of magnitude lower than most Mg2+ content.
Advantages and disadvantages of spin columns:
Efficient and reliable
Produce nucleic acids suitable for downstream applications
Slow-growing strains have low nucleic acid levels
Incomplete elution leads to loss of nucleic acid
The binding capacity of the spin column determines the total amount of nucleic acid
Plasmid Spin Column Kit
Plasmid extraction kits can quickly isolate plasmids from E. coli and are one of the most widely used kits in molecular biology laboratories.
Since E. coli is easy to chemically lyse, the plasmid kit combines sample lysis and nucleic acid purification as follows:
A. Bacterial cells are lysed in a centrifuge tube before the cell debris is centrifuged and precipitated;
B. The configuration of the lysis buffer is such that only the plasmid DNA can be combined with the spin column after lysis. Therefore, the cell lysate can be immediately mounted on the column by centrifugation.
C. Combine the plasmid containing the lysate with the spin column, and the remaining purification steps are similar to all other spin column schemes.
D. Use high-quality plasmid DNA for elution in water or TE buffer.
**Most spin column kits distinguish plasmid DNA from genomic DNA (gDNA) during the lysis process. The lysis buffer contains sodium hydroxide and sodium lauryl sulfate to completely denature the plasmid and gDNA. In the next step, the sample is neutralized and the plasmid DNA is reborn. However, high-molecular-weight gDNA cannot be completely recombined, and is easily entangled with the protein in the sample, thereby preventing the gDNA from binding to the spin column, leading to its removal. Although in terms of efficiency and reliability, plasmid spin column kits have some shortcomings. The optimization standard of the kit is mainly through Escherichia coli strains, slow or unstable strains may have low yields. In addition, plasmid kits and spin column kits have limited binding capacity. Depending on the kit, the total recovery rate is between 50 and 100 μg.
Other spin column kits
In addition to nucleic acid extraction, spin columns are also used for nucleic acid purification and concentration. Purification kits can be used to remove unreacted residual reagents in PCR or other enzyme preparation reactions, and to purify DNA from agarose gels. Concentrated samples can provide greater flexibility for many downstream applications.
Some commercial spin column kits have the function of fragment screening, allowing the selection of suitable fragment range kits (for example, small RNA) according to the research purpose. This is achieved by changing the ethanol content in the buffer solution.
Many spin column kits combine cell lysis and nucleic acid purification. We can find spin columns for nucleic acid separation from almost any type of sample, including but not limited to insects, plants, seeds, fungi, bacteria, saliva, blood, feces and Embed samples in paraffin.
② Phenol/chloroform and ethanol precipitation
The phenol/chloroform extraction method is a method that relies on the principle of different solubility to separate nucleic acids. The sample is exposed to a given ratio of phenol/chloroform mixture to obtain the required nucleic acid. Protein is soluble with phenol/chloroform, nucleic acid is soluble in water. When the phenol/chloroform solution is mixed with the sample, the protein and nucleic acid are separated, providing a guarantee for purification.
For the dual extraction of RNA and DNA, this process can be adjusted by adding acidic phenol. This allows excess H+ ions to interact with the phosphate backbone, resulting in uncharged DNA. DNA will be dissolved in the phenol layer extracted by phenol/chloroform, while RNA will remain dissolved in the water phase due to its natural acidity. After centrifugation, the aqueous and organic phases can be separated, and each phase can be ethanol-precipitated.
Operation steps of classic phenol/chloroform and ethanol precipitation:
A. Contact of phenol and chloroform:
The lysed sample is usually mixed with phenol/chloroform by strong vortexing. The vortex ensures that all organic components can fully interact with the phenol/chloroform mixture to achieve complete dissolution and removal.
B. Centrifugation: After step A, two phases are visible. The aqueous phase containing nucleic acids is at the top, while the organic phase at the bottom contains proteins, lipids and other macromolecules. Then centrifuge the sample to completely separate the two phases
C. Phase separation: carefully remove the water phase with a pipette.
D. Ethanol precipitation: precipitation and purification of nucleic acid with ethanol. During the ethanol precipitation process, salt and ethanol are added to buffer the nucleic acid in the aqueous solution. Salt buffers the sugar phosphate backbone, and ethanol changes the dielectric constant of the solution. This allows the nucleic acid to be separated from the aqueous solution, which can be separated by high-speed centrifugation.
E. Resuspension: Re-dissolve the DNA or RNA in a cluster in water or TE buffer.
Advantages and disadvantages of phenol/chloroform extraction:
High efficiency, usually higher than the yield of the spin column; ü Suitable for extracting complete high molecular weight DNA (eg, gDNA);
Samples suspended in complex solutions are usually still suitable for this method, and some volatile compounds can interfere with the spin column matrix;
For fat samples (eg, brain tissue), phenol/chloroform extraction is better than most spin column kits.
If not handled properly, phenol and chloroform are harmful and must be handled with extreme care in a fume hood.
This method is more time-consuming than spin column kits and may result in lower yields.
The trace amounts of phenol and chloroform in the nucleic acid extract will have a negative impact on downstream enzymatic reactions, such as PCR. If this is the case, it needs to be cleared before PCR. Therefore, there are many spin column purification kits.
A little practical experience may be required to safely extract the contaminant-free water phase.
③Automatic methods to increase throughput
According to different nucleic acid applications and sample types, suitable high-throughput extraction methods can be selected. The most commonly used is magnetic bead extraction. In this technique, positively charged magnetic beads are introduced into the sample, and DNA is combined with the positively charged magnetic beads at low pH, and the DNA is released at high pH. The beads can be removed by a magnet, and the pH of the solution can be easily adjusted to separate the desired nucleic acid. This technology is fast and efficient, but the investment in automation equipment can be quite expensive. (The coating material of the magnetic beads is different, and the principle is slightly different.)
d. Key factors and their impact on extraction
When performing nucleic acid extraction or purification, you must pay close attention to some factors, such as pH, salt concentration, temperature, buffer volume, and potential ethanol contamination. Each of these factors will greatly affect the yield, quality and success rate of downstream experiments.
1. Non-optimal pH or salt concentration can change the charge of nucleic acid, causing nucleic acid to fail to bind to the spin column, or causing phenol/chloroform to dissolve nucleic acid by mistake.
2. Incorrect buffer volume can lead to incomplete lysis, neutralization or indirect dilution of the eluted nucleic acid.
3. Ethanol contamination can inhibit downstream enzyme reactions and make the sample float out of the agarose gel.
e. Special circumstances
1. Extraction of high molecular weight DNA
For some applications (such as Southern hybridization and some sequencing processes), it is necessary to extract intact high molecular weight (HMW) DNA. To extract HMWDNA, in addition to the above factors, other factors need to be considered:
HMW-DNA is easily broken during the extraction process. Because shearing forces (vortex, ultrasound, etc.) can cause the decomposition of DNA molecules, resulting in shorter fragments after extraction. To avoid this, apply minimal shear to the sample.
For applications that require visualization of HMWDNA, lysis and extraction can be performed in an agarose gel plug to stabilize the DNA during the entire process (for example, pulsed field gel electrophoresis), where the entire chromosome remains intact and visualized by electrophoresis.
Alkaline lysis often leads to the loss of HMW-DNA, because HMW-DNA will become irreversibly tangled during the denaturation process.
If you use a spin column to extract HMW-DNA, consider the binding capacity of the column matrix. Some columns can only handle fragments of 10-15 kb in size, because larger fragments are difficult to elute from the column due to tight binding. For larger fragments, it may be necessary to consider dedicated columns with high HMW binding, or manual methods such as phenol/chloroform extraction and ethanol precipitation.
In the traditional RNA extraction process, small RNA molecules are often lost, because traditional spin columns usually have a size limit of about 100 base pairs, although the size limit depends largely on the buffer formulation. Small RNA molecules are extremely important for understanding biological functions, so most total RNA extraction and purification kits are optimized to capture these small RNAs.
2. Evaluate the extracted nucleic acid
a. Quantification and quality control
After nucleic acid extraction and purification, it is very important to understand the concentration and quality of the extracted sample. Depending on the purpose of the downstream experiment, fluctuations in these parameters can greatly change the results. For example, if each cDNA synthesis reaction does not use the exact same total RNA, qRT-PCR expression analysis will result in unreliable data. There are many nucleic acid evaluation methods, which differ in time consumption, cost, and accuracy. Finally, the requirements of downstream experiments should be considered when selecting methods.
The following is an overview of some of the most common assessment methods:
1. agarose gel electrophoresis
Agarose gel electrophoresis is based on molecular weight and separates nucleic acids through a solidified gel matrix in the presence of an electric current. The extracted nucleic acid is compared with a parallel molecular weight standard, which contains fragments of known size and concentration.
Advantages: visually inspect DNA quality; complete genomic DNA samples are shown as complete bands; degraded nucleic acids are shown as diffuse; ribosomal RNA is clearly visible; available for most laboratories; visualization of possible contamination (for example, plasmid purification samples RNA is present in).
Disadvantages: Only a rough concentration estimate is provided; it does not show the presence of contaminants, such as salt, in the sample.
2. Capillary Electrophoresis
Capillary electrophoresis, sometimes referred to as a laboratory on a chip, draws samples through a small capillary monitored by a detector, and the computer receives the information and displays it graphically. Capillary electrophoresis is suitable for analyzing fragment size, quantity and overall quality of the sample. This technique is more accurate than traditional agarose electrophoresis, and is usually a method of nucleic acid evaluation before qPCR.
Advantages: accurate analysis of multiple types of nucleic acids; automatic sample loading and quantification are more accurate than the gel method; high sensitivity-small and small samples can be detected and analyzed.
Spectrophotometry is a rapid detection technique that relies on the characteristics of light interacting with the sample for accurate quantification. The sample is loaded into the test instrument (spectrophotometer) with different wavelengths of light passing through them, and then received by the detector. The detector provides information about the quantity and quality of the sample, which is then translated by the computer software. By measuring absorbance at 260nm and 280nm, the ratio of the two indicates the purity of the sample. For pure DNA and RNA, the 260/280 ratio should be between 1.8 and 2.1. Additional measurements can be made at 230nm, and a 230/280nm ratio below 2.0 indicates the presence of organic contaminants.
In recent years, a new type of fluorescence-based spectrophotometric analysis method has been widely used in molecular biology laboratories. This fluorescence-based technology has higher sensitivity than traditional spectrophotometry, and can distinguish DNA and RNA by using fluorescent dyes unique to DNA and RNA.
Advantages: accurate and fast; provide information about the presence of contaminants; most laboratories have spectrophotometers.
Disadvantages: Unable to quantify a single segment; expensive.
b. Improve nucleic acid quality
Despite our great efforts, it is usually necessary to take additional measures to improve the quality of nucleic acids. The following will describe some additional factors that need to be considered when improving the quality of nucleic acids.
1. Use and storage temperature:
Nucleic acids are easily degraded by nucleases (namely RNase and DNase), and low-temperature storage can help inhibit enzyme activity. DNA is inherently more stable than RNA and is more resistant to nuclease activity at higher storage temperatures.
DNA should be stored at -20°C or below, and kept on ice during use. Repeated freezing and thawing may fragment DNA, especially HMW-DNA. Therefore, if HMWDNA is used frequently, it should be stored at 4°C.
RNA is more easily degraded by nucleases and should always be stored at a very low temperature (-20 to -80°C), and only thawed when necessary.
2. Nuclease inhibitor and cleaning solution
The presence of nuclease can quickly degrade nucleic acid samples. It is easy to transfer nucleases from unprotected hands to the work surface; therefore, you should always wear gloves when working with nucleic acids.
The workplace should be kept clean and frequently wiped with ethanol to remove nuclease contamination. There are many commercial products that can prevent RNase contamination. Many researchers also added the ribonuclease inhibitor diethyl pyrocarbonate (DEPC) to water for RNA work. It is also advisable to wash and treat glassware with EDPC before RNA work.
In recent years, many nucleic acid stabilizers have appeared on the market. Unlike the cleaning solutions described above, these reagents are added directly to the intact sample at the collection point to inhibit nucleases and stabilize nucleic acids until extraction. Although most of these reagents are compatible with most downstream extraction kits and applications, some of them need to be removed from intact samples before nucleic acid extraction.
3. Use nuclease-free consumables
When using any nucleic acid, be sure to nuclease any consumables or glassware. Whenever possible, buy nuclease-free tubes and pipette tips with filters. If after analysis, you find that your DNA yield is low and the quality is not ideal, or if the extracted DNA passes your quality assessment but you get unsatisfactory results during the downstream experiment, you need to do some troubleshooting .