Nucleic acid isolation

Background

Effective isolation of nucleic acids (NAs) is important for sensitive and accurate clinical molecular methods that interrogate deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). There are many different techniques and methods (including commercial kits) available for NA isolation, or sample preparation, and the choice often depends on the source of NA and downstream analytical method. The most common procedures in clinical and research laboratories utilize solid-phase NA separation, such as automated bead separation, or centrifugation filter techniques.

Content

Optimal NA isolation includes lysis from diverse sources such as human cells, viruses, bacterial spores, or protozoan oocysts, and purification of DNA or RNA. Techniques involve sample exposure to chemicals, enzymes, or binding matrices that reduce sample volume, variability, and complexity to achieve purity goals. The isolated NA should be efficiently separated from molecules that could act as potential inhibitors of a downstream molecular assay. The best preparation method depends on the requirements for a specific application. Goals may include flexibility for multiple sample types, large batch processing, speed, or high-purity NA. Consistent results for today’s molecular methods can usually be obtained by the right combination of lysis, concentration, purification, and efficiency for NA isolation.

Lysis

Release of NA from cells, nuclei, or organisms is the first step of any NA preparation. Lysis can be induced by chemical, enzymatic, or mechanical means ( Fig. 63.2 ). It is important to select an appropriate lysis technique for the target to ensure that NA is released efficiently. Sometimes lysis is the only step required for NA analysis. Most human cells and many pathogens (especially viruses and gram-negative bacteria) require only simple chemical lysis (alkaline lysis, salt, detergents, or chaotropic agents). Early methods used alkaline lysis of cells to separate NAs from proteins, with chemicals such as sodium hydroxide. Detergents also aid lysis by breaking down membranes.

Enzymatic lysis, with enzymes such as proteinase K, lysozyme, or mutanolysin, can increase lysis efficiency by degrading membrane or capsid proteins or attacking the peptidoglycan layer. Lytic enzymes can be bacteriolytic or yeast lysing, including lysozymes that digest the extensive peptidoglycan layer of some gram-positive bacteria. For gram-negative bacteria, detergents are used first to remove the outer membrane, then enzymes are used to assist in lysis ( Fig. 63.3 ).

Lysis is a bigger challenge when working with complex sample types or a wider variety of pathogens such as bacterial spores or oocysts. Gram-positive bacteria (with a thicker peptidoglycan layer or proteinaceous spore coat), tissues, fungal cells, and protozoan oocysts are particularly resistant to some lysis techniques because of their complex cell walls. More efficient techniques should be used if target NA is low in concentration and requires efficient release of NA in order to be detected. Resistant organisms may require more aggressive chemical, enzymatic, or physical methods. Bacterial spores, fungi, yeasts, and oocysts have complex coats or walls containing proteins and other complex molecules cross-linked to make them resistant to many environmental factors. Given that these organisms have very different molecules that make up their structure, it is difficult to design a universal enzymatic or chemical approach. Mechanical lysis, using external physical force, is a method for many cell types discussed later. Other physical manipulations, such as sonication, or temperature changes, such as boiling ^, or freeze–thaw cycles, can lyse cells.

Lysis by physical means is often the best option for many hardy pathogens that are difficult to disrupt. Physical means are nonspecific methods that work with many sample types. Although it requires specialized equipment, mechanical lysis is being widely adopted because it is a rapid method that can work for many target types. Mechanical lysis systems require a large input of energy and may be loud, but they do not require the addition of chemicals or enzymes that need to be removed later. NA shearing during mechanical lysis can be a concern, so care is taken to minimize lysis time, but shearing may not impact detection if the NA fragments are larger than what is required for analysis. Bead milling is a type of mechanical lysis that involves rapid motion of small beads to physically break open cells. ^, Beads can be moved rapidly in many ways, including movement of the container. Sonication is another mechanical method by which high-intensity sound waves can be applied to lyse cells. If an analysis system targets NA from many types of cells or organisms, a broad approach such as mechanical lysis is desirable. Some automated NA systems have additional external mechanical lysis options. ^,

Protein removal

In a cellular environment, NAs are surrounded by proteins called histones and many other accessory proteins. Proteins can be detrimental to either the NA preparation process or downstream analyses. Endogenous enzymes, such as nucleases, can break down the target NAs during the preparation if not removed, proteases can interfere with downstream enzymatic procedures, and large amounts of proteins can interfere with specific binding of NA in some systems. Therefore it is often important to denature or remove proteins from a sample.

Some samples contain large amounts of RNases, and RNA is particularly unstable in the presence of RNases. Some techniques used to eliminate proteins are effective against RNases, such as proteases or strong denaturing agents. Usually RNases need to be degraded or denatured immediately in the lysis reaction because RNA degradation by RNases can be very rapid. It is also important to use labware that is free from RNases when preparing RNA because RNases are prevalent on skin and in the environment.

Chemical or enzymatic techniques can be used to eliminate proteins by degradation or precipitation. Chaotropic acids such as guanidine hydrochloride or guanidinium thiocyanate protect NAs from nucleases because of their potent protein denaturing properties. Chaotropic acids are also useful because they lyse bacteria and yeast. These chemicals are used in many NA preparation methods.

Detergents are added in many NA preparation methods to dissociate or remove proteins from NA preparations. Sodium dodecyl sulfate (SDS) was an early detergent used in the preparation of NA to help separate NAs from proteins (including nuclear and membrane-bound proteins). Its use evolved from the observation that SDS and other surfactants disrupt bacterial and viral structures by solubilizing the proteins. Another common detergent used is Triton X-100.

Unwanted proteins and enzymes can also be digested by the addition of proteases in the method. Proteases, which have many purposes, including protection from infection, are naturally occurring enzymes found in plants, animals, and microorganisms. Some proteases are used in NA preparations to reduce protein background and aid in lysis by digesting membrane or capsid proteins. Chemicals and detergents can only denature proteins, but proteases actually break them into smaller molecules by cleaving peptide bonds. Most proteases, however, are too specific in their cleavage sites and not useful for NA preparations or are too difficult to produce in production quantities. An exception is the primary protease used for NA preparations, the serine protease proteinase K, originally isolated from Engyodontium album . Its broad lysis specificity and protein degrading properties are very useful for NA preparations. Other proteases, such as the temperature-stable proteinase EA1, are also used in NA preparations. Proteolysis may require an incubation step at 37 to 55 °C, and the enzymes need to be removed or inactivated because they often interfere with downstream analysis.

Separation techniques

When cell lysis is complete, NA can be isolated from other cellular or sample materials by separation methods. Proteins, polysaccharides, metals, salts, organic compounds, and dyes are examples of molecules that may need to be removed. Isolation is not required if molecules are tolerated or if a sample is relatively clean. Several techniques have been used to isolate NA from other components of the cell or sample background. The following paragraphs discuss separation methods in two broad categories: (1) liquid–liquid extraction (liquid phase separation and precipitation) and (2) liquid–solid extraction (by size exclusion or affinity separation) ( Fig. 63.4 ). The process of isolation can also concentrate the NA, increasing the sensitivity of downstream detection methods.

Liquid phase extraction is a common method used for NA isolation that leads to a pure product. NA can be isolated from other molecules by differential solubility in immiscible liquids ( Fig. 63.5 A). The primary organic solvent is phenol, usually mixed with chloroform and isoamyl alcohol. Phenol denatures proteins, which stay in the organic phase while the NA is in the aqueous phase. The addition of chloroform and isoamyl alcohol helps to separate the phases and prevent foaming. After separation, NA in the aqueous phase is precipitated by ethanol to remove residual phenol for a clean, concentrated product. Initially designed to purify relatively unstable RNA vulnerable to degradation by RNases, the phenol–chloroform extraction method is very effective but also manually tedious, must be performed in a fume hood, and creates hazardous waste. Although not currently well suited for high-throughput needs, microfluidic liquid phase extraction holds promise because liquids are easy to manipulate.

Precipitation of NA is a liquid phase method that achieves a clean product and concentrates NA. NA precipitates in the presence of alcohols, such as ethanol or isopropanol, and a high concentration of salt (0.1 to 0.5 M), such as ammonium acetate, sodium acetate, or sodium chloride. Other liquids that can precipitate NA are acetone and lithium chloride. Centrifugation concentrates a NA pellet from the rest of the liquid, and the pellet is manually dried and resuspended. The resulting NA is clean, although some molecules can co-precipitate with the NA. Because manipulation of the pellet is required, this method is tedious but results in less hazardous waste than phenol–chloroform extraction.

Solid phase extraction methods are the most common method of NA isolation for several reasons, including minimal use of hazardous chemicals, fewer and easier manual manipulations, easy adaptation to automation, and increased throughput. Solid phase extraction is often described in four basic steps: lysis, binding, washing, and eluting.

Solid phase methods rely on three principal techniques: size exclusion by gel filtration, ion exchange chromatography by charge-based reversible adsorption, and affinity chromatography by reversible surface adsorption. Any of these methods can be incorporated into spin filters, columns, or beads.

In gel filtration, NA molecules can be separated from smaller molecules by size through a gel matrix (see Fig. 63.5 B). Using this method, the gel matrix allows larger molecules to pass through while smaller molecules are delayed within the pores. This is useful for separating NA from large molecules, but molecules similar in size will separate with the NA. Sephadex or derivatives are the most common matrices used. Electrophoresis or isotachophoresis methods allow NA to be separated from other molecules by moving through the gel matrix through an electric field. A similar method moves NA-bound beads through an immiscible liquid wash phase to remove contaminants.

In ion exchange chromatography, negatively charged NA molecules can bind selectively to surfaces with charged groups surrounded by free counter-ions (see Fig. 63.5 C). Charged NA exchange places with the ions and bind to the surface by charge. Unbound substances are washed away. NA is released by displacing it with a flood of free ions that replace the NA molecules. For example, diethylaminoethyl cellulose (DEAE-C) is a common anion exchange resin that negatively charged NA will bind to. NA is released when other ions in a high salt buffer are present to exchange with the NA. Ion exchange techniques can also be used in reverse to specifically bind and separate unwanted molecules. For example, Chelex resin (Bio-Rad Laboratories) is used to separate metallic compounds and inhibitors of PCR away from NA.

Affinity chromatography, using reversible adsorption of NA to surfaces (see Fig. 63.5 D) such as silica, is the separation of choice for many NA preparation procedures. This technique is commonly used in automated methods. All NAs will bind to silica surfaces under specific binding conditions, especially in the presence of chaotropic salts ( Fig. 63.6 ). Binding occurs when linear NA adsorbs lengthwise to silica surfaces because of complex hydrogen bond formation between the silica and NA surfaces in the presence of chaotropic salts or alcohols at high concentration and low pH (below pH 7). Because both silica and NA surfaces are negatively charged, the binding is due to adsorption in high ionic strength conditions and hydrogen bonding that occurs as water is removed from the surfaces. The NA is released when the salt or alcohol is removed and the surfaces are hydrated. Any surface with similar NA binding properties can be used in this way, such as diatomaceous earth. Serendipitously, similar chemicals can be used for lysis and surface binding. Chaotropic salts can be used for cell lysis and binding to a silica surface. In the 1990s, NA preparation became simplified using this method. Washing of the silica surface is often accomplished with alcohol. Elution occurs when binding to silica is reversed with water. Elution with small volumes can help increase the concentration of target. Unlike ion exchange, no specific chemical is required for elution using affinity chromatography.

Solid affinity isolation is flexible in that the binding surface can be created on a variety of solid surfaces. A binding filter or column is commonly used. A liquid sample passes through by centrifugation (spin filter), pressure (syringe filter), or vacuum. Kits with silica spin filters are quick to perform and do not require hazardous chemicals. A drawback is that filters can clog with thick sample types and steps are necessary to load the binding, washing, and eluting solutions. The binding surface can also be on beads or particles that mix freely with a sample to collect free NA. Glass beads or particles are the simplest silica-based surfaces. Because the beads move through the sample, clogging is not a concern. Beads or particles can then be collected by filtration of particles, centrifugation, or a magnet. Surface binding capacity is determined by the area available for binding.

Surface binding methods have been improved extensively for automation and simplicity. Paramagnetic beads coated with NA binding surfaces such as silica are used widely on automated platforms. ^, Paramagnetic beads do not generate a magnetic field themselves but respond to external magnetic forces; this property of paramagnetic beads is used to move the beads through solutions. Many silica paramagnetic systems are commercially available and are amenable to the use of aqueous chemistry.

Paper surface binding methods, in which NAs bind to cellulose, are convenient and fast. Chemically treated paper contains lysis and binding reagents that combine when the sample is applied. Lysis and binding occur in the paper, followed by washing and elution off the paper, usually in a small volume.

Isolating DNA versus RNA

DNA and RNA may be co-prepared from the same sample. Some molecular analysis methods may target both DNA and RNA, and preparation of both is desired. Many methods will prepare both DNA and RNA from the same sample, although special effort must go into protecting RNA, if desired, from degradation, especially from RNases. Chaotropic agents are effective at removing nucleases, including RNases.

Unwanted NA, such as background RNA or single-stranded DNA (ssDNA) can be enzymatically or chemically removed from a preparation. Some methods are designed for only DNA or RNA and contain steps to remove the other NA, usually by adding a nuclease specific for the undesired NA. Specific NA can also be separated by size exclusion or with liquid phase separation techniques such as phenol–chloroform.

Nucleic acid concentration

Sometimes NAs are present at very low concentrations in samples. Various techniques can be used for increasing the target NA concentration during preparation. The target can be concentrated before or after lysis by either isolating target cells or by concentrating all NA during preparation.

There are a variety of techniques that can be employed to increase target cell concentration prior to NA isolation. Cell growth or selection can lead to increased concentrations. Some pathogens can be grown to higher quantities for detection by culture, but this requires days to weeks, depending on the organism. Selective recovery of cells by centrifugation or filtration is sometimes possible, but in complex samples, too much unwanted material can clog a filter or overwhelm the system. Centrifugation methods are often used for blood. For example, red blood cells (RBCs) can be specifically lysed to allow recovery of other cells, or RBCs containing malaria can be selectively recovered by density gradient centrifugation. White blood cells (WBCs) can be concentrated by centrifuging anticoagulated blood, resulting in a layer enriched in WBCs (the “buffy coat”) between the RBCs and plasma. Selection by binding is another concentration method. Some paramagnetic beads used for NA isolation also may bind to bacterial cells nonspecifically. Beads with specific antibodies are used in immuno-magnetic separation techniques designed to bind specific bacteria or cells as a concentration method. New methods have been developed to selectively concentrate circulating cancer cells, and these techniques are covered in Chapter 71 .

Nucleic acid precipitation

The prepared NA product can also be concentrated after extraction if a large volume of material is produced. Depending on how the NA was extracted, several subsequent cycles of extraction with butanol or centrifugation can be performed to concentrate the NA. Alcohols such as ethanol or isopropanol can be used to precipitate NAs in the presence of high salts. Isopropanol is less volatile than ethanol, but some salts are less soluble in isopropanol compared to ethanol, so extra washings may be required when using isopropanol. Precipitate can be collected by centrifugation and washed to remove salt for concentrated NAs. Binding columns or dialysis can also be used to remove water and concentrate molecules in solution.