Know-How for Nucleic Acid Extraction and Purification

Transcription

1 Contact The Bitesize Book of Know-How for Nucleic Acid Extraction and Purification

2 Table of Contents 1. An Introduction to Nucleic Acid Extraction and Purification Why Isolate Nucleic Acids? Cell Lysis Methods Underlying Principles of Nucleic Acid Purification 1.4 Critical Factors and How They Influence Success Special Situations Assessing Your Isolated Nucleic Acids 2.1 Quantification and Quality Control 2.2 Improving Nucleic Acid Quality

3 1. An Introduction to Nucleic Acid Extraction and Purification Putting science first New England Biolabs Over 1,000 articles have been published by NEB scientists to date 283 restriction enzymes are supplied by NEB, >210 of which are active in CutSmart Buffer Nucleic acid extraction is a fundamental part of molecular biology because high quality nucleic acids are critical to most applications. In this guide, we will provide an overview of the currently used techniques for nucleic acid isolation and some tips to help you choose the right technique for your sample type. We will cover the ways in which you can assess purified nucleic acids for quantity and quality, and we will introduce the most commonly encountered problems during nucleic acid extraction. New England Biolabs is a recognized world leader in the discovery, development, and commercialization of recombinant and native enzymes for genomic research. Page - 3

4 1.1 Why Isolate Nucleic Acids? Isolated nucleic acids provide answers to a plethora of research questions across a broad range of applications (e.g., cloning, qrt-pcr, and genome- or transcriptomewide next generation sequencing). The information obtained from isolated nucleic acids can be used in a number of ways. The exact research goal determines the type of nucleic acid to be extracted and the application often influences the choice of extraction method. For example, a standard end-point PCR reaction does not require the same DNA quality as a whole-genome sequencing experiment. To identify the best extraction method for your work, it is necessary to have a clear understanding of the downstream application, as well as any potential limitations associated with your sample type (e.g., clinical samples are often limited in amount and challenging to work with). Depending on the sample type, the cell lysis method may differ, but the overall nucleic acid extraction concept will remain the same: cells or tissue samples are lysed and non-nucleic contaminants (e.g., proteins) are removed, before the nucleic acids are washed and concentrated. Reasons for isolating nucleic acids 1. To analyze gene expression for basic or disease-oriented research. 2. To follow responses to medical treatments (e.g., monitoring of viral titres during and after anti-viral therapy). 3. To identify new species and to gain a deeper understanding of evolutionary processes (e.g., ancient DNA analysis). 4. To monitor and type pathogens responsible for infectious disease outbreaks in humans, animals, and plants. 5. To monitor food and water safety via microorganism detection and quantification. 6. To diagnose diseases (e.g., genetic disorders, cancer, immunological deficiencies). Page - 4

5 1.2 Cell Lysis Methods The cell lysis method used depends to a large extent on the sample type, with tougher tissues like plants requiring more force than mammalian cells. Info An overview of the most commonly used lysis methods for a range of sample types is presented in Table 1. Cell lysis methods are grouped into 3 main categories: mechanical, enzymatic, and chemical lysis. The principles underlying the 3 lysis approaches and the pros and cons of each are presented below: Mechanical Lysis In mechanical lysis, cellular membranes are disrupted by an external force (see Table 1 for examples). Pros Often efficient and fast Fast lysis reduces time between harvesting the sample and the isolation of nucleic acids, which might be crucial in gene expression analysis experiments Ideal for tough-to-lyse samples (e.g., plant material, filamentous fungi, and yeast) Cons Time-consuming when processing multiple samples, depending on method used (e.g., pestle and mortal processing is very slow) Slow processing of large sample numbers may increase the risk of sample degradation Can generate heat in a sample, leading to protein aggregation and nucleic acid degradation Requires special equipment/tools Page - 5

6 1.2.2 Enzymatic Lysis In enzymatic lysis, an enzyme is added to digest proteins or cellular structures, such as cell walls of yeast and filamentous fungi. Pros Ideal in a lab that doesn t possess mechanical lysis equipment Selective removal of the cell wall leaves behind the remainder of the cell for another lysis method (usually chemical). This provides flexibility and circumvents some of the damage that may be caused by mechanical lysis Chemical Lysis During chemical lysis, cells are washed with a detergent that breaks down lipid membranes, thus releasing cellular components. In addition to a detergent, chemical lysis buffers usually contain chaotropic salts, such as guanidine hydrochloric acid or urea, which help destabilize proteins (e.g., nucleases) that might degrade the newly exposed nucleic acids, and prime the nucleic acid for binding to silica-matrixes. The exact composition of a chemical lysis buffer varies depending on the application and sample type. Cons Can be time-consuming (enzymatic digestion may take 1 hour) and expensive Not ideal for gene expression analysis as enzyme-induced cellular changes may impact gene expression Next Page Table 1: Overview of Commonly Used Lysis Methods. Note: It is not possible to list every sample type here, so if your sample type doesn t appear in the table, consult your colleagues or suppliers to find the correct lysis method. Pros Cheap, easy, fast to perform No specialized equipment required Cons Detergents that disrupt the cell membrane will often lyse other intercellular membranes as well, and thereby releasing their components. This approach isn t readily suitable for organelle-specific nucleic acid extraction While this technique works well for E. coli, it is not effective for gram-positive bacteria, plant cells, or fungal cells because of the presence of hard cell walls that prevent the detergent from accessing the cell membrane Adding both a detergent and chaotropic salt to an E. coli sample may compromise the ability to differentiate plasmid DNA from genomic DNA. However, steps can be taken to differentiate these, which will be discussed later Harsh chemicals can present a danger to the researcher Page - 6

7 Method Waring Blender Category 1: Mechanical Lysis Description Blender blades grind samples at high speed Sample Type Yeast, complex tissue samples (e.g., liver, muscle) Dounce Homogenizer Grinds samples while leaving organelles intact Cultured cells Bead Beater Metal beads are agitated with samples at high speed Mammalian, plant, microorganisms Manual grinding with mortar and pestle Samples are ground with a mortar and pestle in liquid nitrogen Plant, filamentous fungi Freeze-Thaw Samples are subjected to multiple freeze/thaw cycles Bacteria French Press Sonication Method Lysostaphin Glucanase, chitinase Samples are disrupted as they are forced through a small space Samples are broken down with high frequency sound waves Category 2: Enzymatic Lysis Description Enzymes that target glucan and chitin, respectively, in fungal cell walls Targets glucan and chitin, respectively, in fungal cell walls Plant, filamentous fungi All samples types Sample Type Staphlococcus spp. Yeast, filamentous fungi Cellulase Macerozyme Enzyme that breaks down cellulose, a major component of plant cell walls Enzyme used in conjunction with cellulase to break down plant cell walls Mammalian, plant, microorganisms Plant Freeze-Thaw Method Alkaline Lysis Page - 7 Samples are subjected to multiple freeze/thaw cycles Category 3: Chemical Lysis Description Series of chemical washes to extract nucleic acids from bacterial cells Plant Sample Type Bacterial plasmids

8 1.3 Underlying Principles of Nucleic Acid Purification After cell lysis, nucleic acids are selectively isolated from the surround cellular components, concentrate, and stored in water or a suitable buffer for use in downstream applications. The steps following cell lysis are collectively referred to as purification. Since the chemistry of the isolated nucleic acid remains the same regardless of sample type, the purification strategies described below are generally applicable to all sample types Spin Columns Often referred to as using the kit, spin column extraction and purification allows rapid purification and cleanup of high quality genomic DNA, plasmids, RNA, and PCR products, without the need to make fresh solutions. Spin columns contain a solid matrix of silica or proprietary resin for selective binding of the nucleic acid of interest. A Typical Spin Column Protocol: 1. Application of lysed sample to spin column The lysed sample, collected in a binding buffer containing chaotropic salts, is applied to a spin column. The binding buffer allows the nucleic acids to disassociate from the aqueous solution and bind to the column matrix. 2. Nucleic acid binding 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 often called the flow-through). 3. Washing After binding, the column is washed to remove any remaining contaminants like protein or residual salts that may severely impair downstream applications. The number of washes performed is kit-dependent. Some wash buffers contain chaotropic salts to remove protein, and some have a high ethanol concentration for salt removal. Most kits include a final wash step in a buffer almost entirely comprised of ethanol to ensure complete salt removal. 4. Spin to removal residual ethanol After washing, a short dry spin is sometimes performed to remove Page - 8

9 residual ethanol. 5. Elution To elute the nucleic acids from the matrix, a small volume of water or elution buffer* is added and the column is allowed to rest for a few minutes. Since chaotropic salts have been removed in Step 4, the aqueous elution buffer will now be able to rehydrate the nucleic acids, disassociating them from the column matrix. A final spin allows this aqueous solution containing the nucleic acid sample to be transferred to a fresh tube. * Nucleic acids are usually stored in a buffer containing Tris and EDTA (TE buffer). The presence of EDTA helps to inhibit nuclease activity. While TE buffer is effective in inhibiting nucleases, the amount of EDTA present in TE is generally orders of magnitude lower than the amount of Mg2+ present in most enzymatic reactions, so the presence of EDTA in the elution buffer should not be a concern. Pros and Cons of spin column kits Pros Cost effective High efficiency Reliable Yields nucleic acids suitable for use in downstream applications Cons Slow growing strains can produce lower yield Nucleic acid loss caused by incomplete elution Yield is limited by binding capacity of column Page - 9

10 Nucleic Acid Estraction and Purification 3. After binding of the plasmid-containing lysate to the spin column, the remaining purification steps are similar to all other spin column protocols. 4. Ready-to-use high quality plasmid DNA is eluted in water or TE buffer A Note on Plasmid Spin Kits Plasmid miniprep kits are among the most widely used kits in molecular biology labs, allowing for rapid plasmid isolation from E. coli. Since E. coli is amenable to chemical lysis, plasmid kits combine sample lysis and nucleic acid purification as follows: 1. Bacterial cells are pelleted and lysed in microcentrifuge tubes, before the cellular debris is pelleted by centrifugation. 2. The lysis solution is formulated so that only the plasmid DNA binds to the spin column after lysis**. Therefore, the cell lysate can be loaded onto a column immediately following centrifugation. Page - 10 **Most spin kits differentiate plasmid DNA from genomic DNA (gdna) during the lysis step. The lysis buffer contains sodium hydroxide and sodium dodecyl sulfate, which completely denature plasmid and gdna. The proceeding step neutralizes the sample, allowing the plasmid DNA to reanneal. High molecular weight gdna, however, cannot fully reanneal and tends to tangle with proteins present in the sample, preventing the gdna from binding the spin column, thus leading to its removal. Despite their efficiency and reliability, plasmid spin kits come with a few drawbacks. Since they are optimized for use with standard E. coli strains, slow growing or unstable strains will likely produce low yields if used with standard protocols. Furthermore, plasmid miniprep kits, and spin kits in general, are limited in binding capacity, with a total yield of between 50 to 100 μg, depending on the kit. Also, the DNA isolated with these kits generally has higher endotoxin levels. For routine transfections where larger yields and endotoxonfree DNA are required, it is worth considering a midi- or maxiprep kit, which use spin columns with larger loading and binding capacities.

11 Other Spin Column Kits In addition to nucleic acid extraction, spin columns are also used for nucleic acid cleanup and concentration. Cleanup kits can be used to remove unused PCR reagents following PCR and other enzymatic reactions, and to purify DNA from agarose gels. Concentrating your sample can provide more flexibility with many downstream applications. Some commercial spin column kits have size-selection capabilities, allowing you to select the fragment size range you want to study (e.g., small RNA species). This is accomplished by altering the amount of alcohol present in the buffer system. Many modern spin kits combine cell lysis and nucleic acid purification, and nowadays you can find a spin column for nucleic acid isolation from almost any sample type, including, but not limited to, insects, plants, seeds, fungi, bacteria, saliva, blood, feces, and FFPE-samples. applications. Finally, Monarch kits allow small volume elution, resulting in more concentrated nucleic acids and eliminating the need for downstream concentration steps. NEB has recently released the Monarch line of nucleic acid purification kits. This line of spin column kits is specifically designed to reduce environmental impact by including thin-walled columns, which contain reduced amounts of plastic. Additionally, these kits ensure less buffer retention, resulting in higher purity nucleic acids, for greater results in downstream Page - 11

12 1.3.2 Phenol/Chloroform and Ethanol Precipitation Phenol/chloroform extraction is a manual method that relies on the principle of differential solubility to isolate nucleic acids. Samples are exposed to a mixture of phenol and chloroform in a given ratio, depending on the desired nucleic acid. Protein is soluble in phenol/ chloroform, and nucleic acids are water-soluble. When the phenol/chloroform solution is mixed with the sample, proteins and nucleic acids are separated, paving the way for purification. For dual extraction of RNA and DNA, this procedure can be adjusted by the addition of acid phenol. This renders DNA uncharged as a result of the excess H+ ions interacting with the phosphate backbone. DNA will then be soluble in the phenol layer of the phenol/chloroform extraction, while RNA will remain soluble in the aqueous phase, owing to its naturally more acidic nature. The aqueous and organic phases can be separated after centrifugation and each can be subjected to ethanol precipitation, as described above. A Typical Phenol/Chloroform Extraction Protocol: 1. Exposure to phenol and chloroform: Previously lysed samples are mixed with phenol/chloroform, usually by strong vortexing. Vortexing ensures that all organic components can fully interact with the phenol/chloroform mixture for complete solubilization and removal. 2. Centrifugation: After Step 1, two phases are visible. The aqueous phase containing the nucleic acids sits at the top, while the organic phase on the bottom contains proteins, lipids, and other macromolecules. The sample is then centrifuged to fully separate the two phases. 3. Phase separation: The aqueous phase is carefully removed by pipetting. 4. Ethanol precipitation: The nucleic acids are purified via ethanol precipitation. During ethanol precipitation, salts and ethanol are added to buffer the nucleic acids from the water solution. The salts buffer the sugar phosphate backbone, and the ethanol alters the solution s dielectric constant. This allows the nucleic acids to separate from the aqueous solution, permitting subsequent isolation by high-speed centrifugation. 5. Resuspension: Pelleted DNA or RNA is resuspended in water or TE buffer. Page - 12

13 Pros and Cons of phenol/chloroform extraction: Pros Cons High efficiency, often resulting in higher yield than spin kits Suitable for extraction of intact high molecular weight DNA (e.g., gdna) Samples that are suspended in complex solutions are usually still amenable to the procedure, whereas some volatile compounds can interfere with a spin column matrix For fatty samples (e.g., brain tissue), phenol/ chloroform extraction is superior over most spin column kits Phenol and chloroform are harmful if handled incorrectly, and must be treated with extreme caution in fume hoods, as well as be disposed of in appropriate waste streams This method is much more time-consuming than a spin kit, and may result in a lower yield Traces of phenol and chloroform in resulting nucleic acid extracts can negatively impact downstream enzymatic reactions, like PCR. If this is the case, the nucleic acids will need to be cleaned up prior to PCR. A number of spin column PCR cleanup kits exist for this purpose It can take a bit of practice to be able to confidently extract the aqueous phase without contaminants Automated Methods for Higher Throughput Depending on the application and sample type, there may be high-throughput techniques available for your application. One of the most commonly used high-throughput techniques is the use of magnetic bead separation. In this technique, positively charged magnetic beads are introduced to the sample. DNA will bind to the positively charged beads at low ph and will be released at high ph. Beads can be removed through the use of magnets, and the ph of the solution can be easily adjusted to isolate the desired nucleic acid. The technique is fast and efficient, but the investment in automation equipment can be quite costly. Page - 13

14 Nucleic Acid Estraction and Purification 1.4 Critical Factors and How They Influence Success When performing nucleic acid extraction or purification, it is important to pay close attention to a number of factors such as ph, salt concentration, temperature, buffer volume, and potential ethanol contamination. Each of these factors can greatly impact yield, quality, and Suboptimal ph or salt concentration can alter the charge of the nucleic acid in question, which could prevent binding to a spin column, or lead to solubility in the wrong phase of a phenol/chloroform reaction. success of downstream applications. Incorrect buffer volumes can lead to incomplete lysis, neutralization, or undesired dilution of the eluted nucleic acids Ethanol contamination can inhibit downstream enzymatic reactions and cause your sample to float out of an agarose gel well. Page - 14

15 1.5 Special Situations High Molecular Weight DNA Extraction For some applications (e.g., southern blotting and some sequencing procedures), extraction of intact high molecular weight (HMW) DNA is necessary. To extract HMW DNA, additional factors need to be considered in addition to the ones described above: HMW DNA can easily fracture during extraction. Exposure to shearing forces (vortexing, sonication, etc.) can lead to the breakdown of DNA molecules, resulting in shorter fragments after extraction. To avoid this, use an extraction and lysis protocol that places minimal force on the sample. For applications in which visualization of HMW DNA is necessary, it is possible to perform lysis and extraction within agarose gel plugs, thus stabilizing the DNA throughout the procedure (e.g., pulsed-field gel electrophoresis), where entire chromosomes are kept intact and visualized by electrophoresis. DNA because it becomes irreversibly tangled during the denaturation step. If using a spin column for HMW DNA extraction, consider the binding size capacity of the column matrix. Some columns are only able to reproducibly purify kb sized fragments because larger fragments are difficult to elute from the column due to tight binding. For larger fragments, it may be necessary to consider specialized columns for high HMW binding, or a manual method such as phenol/chloroform extraction and ethanol precipitation Small RNAs Small RNA molecules are often lost during traditional RNA extraction procedures because traditional spin columns usually have a lower size limit of approximately 100 base pairs, though size cut-offs are very dependent on buffer formulation. Because small RNA molecules are extremely important for understanding biological function, most modern clean up kits for total RNA extraction are optimized to capture these small RNAs. Alkaline lysis often results in the loss of HMW Page - 15

16 2. Assessing Your Isolated Nucleic Acids 2.1 Quantification and Quality Control Following nucleic acid extraction and purification, it is important to understand the quantity and quality of the extracted samples. Depending on the downstream application, fluctuations in these parameters can drastically alter the outcome. For example, expression analysis by qrt-pcr will result in unreliable data if the exact same amounts of total RNA are not used for each cdna synthesis reaction. There are a number of nucleic acid assessment methods, which vary in time consumption, cost, and precision. Ultimately, the method should be chosen with the requirements of the downstream application in mind. Here is an overview of some of the most common assessment methods: Page - 16

17 2.1.1 Agarose Gel Electrophoresis Agarose gel electrophoresis separates nucleic acids based on their molecular weight by pulling them through a solidified gel matrix in the presence of an electric current. Extracted nucleic acids are compared to a molecular weight standard run in parallel, containing fragments of known sizes and quantity. Pros Visual determination of DNA quality Intact genomic DNA samples appear as bands that barely migrate out of the well Degraded nucleic acid appears as a smear Ribosomal RNA can be clearly seen Available to most laboratories Allows you to visualize the presence of other potential nucleic acid contaminants (e.g., the presence of RNA in plasmid purification samples) Cons Provides only a crude estimate of quantity Does not reveal the presence of contaminants, such as salts, within a sample Page - 17

18 Nucleic Acid Estraction and Purification Capillary Electrophoresis Capillary electrophoresis, sometimes referred to as lab-on-a-chip, pulls samples through a small capillary that is monitored by a detector. A computer receives the information and displays it graphically. Capillary electrophoresis is suitable for analysis of fragment size, quantity, and overall sample quality. This technology is much more precise than traditional agarose electrophoresis, and is often the method of choice for nucleic acid assessment prior to qpcr. Pros Cons Precise analysis of most types of nucleic acids Automated sizing and quantification is much more exact than gel-based methods High sensitivity can detect and analyze even small amounts of sample Page - 18 Expensive

19 2.1.3 Spectrophotometry Spectrophotometry is a fast technique that relies on the properties of light interacting with samples for precise quantification. Samples are loaded into an instrument (the spectrophotometer) that passes light at varying wavelengths through them, which is then picked up by a detector. The detector provides information regarding sample quantity and quality that is then translated via computer software. Absorbance measurements are taken at 260 nm and 280 nm, and 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. An additional measurement can be taken at 230 nm, and a 230/280 nm ratio below 2.0 indicates the presence of organic contaminants. Recently, a new type of fluorescence-based spectrophotometric analysis has become prevalent in molecular biology labs. This fluorescencebased technique achieves increased sensitivity over traditional spectrophotometry, and is able to distinguish between DNA and RNA via the use of DNA- and RNA-specific fluorescent dyes. Pros: Accurate and fast Provides information on the presence of contaminants Spectrophotometer available to most laboratories Cons: Inability to quantify individual fragments Expensive Page - 19

20 Nucleic Acid Estraction and Purification 2.2 Improving Nucleic Acid Quality Despite our best efforts, it is often necessary to take additional steps to improve nucleic acid quality. The following sections will describe some additional factors to consider when improvements in nucleic acid quality are needed Working and Storage Temperature Nucleic acids are susceptible to degradation by nucleases (i.e., RNases and DNases), and storage at low temperatures helps inhibit this activity. DNA is inherently more stable than RNA, and is more resistant to nuclease activity at higher storage temperatures. DNA should be stored at or below -20 C, and kept on ice while in use. Bear in mind that repeated freeze-thaw cycles may fragment DNA, especially HMW species. HMW DNA extracts should therefore be stored at 4 C if they are used frequently. RNA is much more susceptible to nuclease degradation and should always be stored at very low temperatures (-20 to -80 C), and only thawed when absolutely necessary. Page Nuclease Inhibitors and Cleaning Solutions As mentioned previously, the presence of nucleases can quickly degrade a nucleic acid sample. It is easy to transfer nucleases from an ungloved hand to a work surface; thus, gloves should always be worn when working with nucleic acids. Workspaces should be kept clean and frequently wiped down with ethanol to remove nuclease contamination. There are a number of commercially available products to prevent RNase contamination. Many researchers also add the RNase-inhibitor diethyl pyrocarbonate (DEPC) to water that is destined for RNA work. It is also advisable to wash and treat glassware with DEPC prior to RNA work. In recent years, a number of nucleic acid stabilizing reagents have appeared on the market. Unlike the cleaning solutions described above, these reagents are added directly to the intact sample at the point of collection to inhibit nucleases and stabilize nucleic acids until extraction is carried out. While the majority of these reagents are compatible with most downstream extraction kits and applications, bear in mind that some of them require removal from the intact sample prior to nucleic acid extraction.

21 encountered issues during nucleic acid purification. The tips in NEB s guide may also be useful for kits from other commercial suppliers, but it is advisable to consult the supplier of your kit for the most precise troubleshooting advice. You may also find it useful to read protocol-based scientific articles. The underlying cause of suboptimal nucleic acid extraction is often resolved easily when you fully understand the underlying principles of the extraction technique Use Nuclease-Free Consumables When working with any nucleic acid, ensure that any consumables or glassware are treated for nucleases. Purchase nuclease-free tubes and pipette tips with filters when possible. If, following analysis, you find that your DNA yield is poor, the quality is suboptimal, or if the isolated DNA passes your quality assessments but you achieve suboptimal results in downstream applications, you will need to do some troubleshooting. If you ve followed the troubleshooting advice from your kit s supplier, and you can t rectify the issue, then it may be time to contact technical support. Technical support representatives are very knowledgeable and are likely to have encountered other users of your kit with similar issues. Because there are many potential pitfalls, there are also many troubleshooting steps. NEB s troubleshooting guide for nucleic acid purification covers the many commonly Page - 21

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