Isolation of DNA
Preparing The Sample
Nucleic acid is routinely isolated from human, fungal, bacterial, and viral sources in the clinical laboratory. The initial steps in nucleic acid isolation depends on the nature of the starting material.
Nucleated Cells in Suspension
Depending on the type of clinical sample that is sent for analysis, the specimen may have to be pretreated to make nucleated cells available from which the nucleic acid will be extracted. For instance, white blood cells (WBCs) must be isolated from blood or bone marrow specimens.
This is done by either differential density gradient centrifugation or differential lysis. For differential density gradient centrifugation, whole blood or bone marrow mixed with isotonic saline is overlaid with Ficoll.
Ficoll is a highly branched sucrose polymer that does not penetrate biological membranes. Upon centrifugation, the mononuclear WBCs (the desired cells for isolation of nucleic acid) settle into a layer in the Ficoll gradient that is below the less dense plasma components and above the polymorphonuclear cells and red blood cells (RBCs). The layer containing the mononuclear cells is removed from the tube and washed by at least two rounds of resuspension and centrifugation in saline before proceeding with the nucleic acid isolation procedure.
Another method used to isolate nucleated cells takes advantage of the differences in the osmotic fragility of RBCs and WBCs. Incubation of whole blood or bone marrow in hypotonic buffer or water will result in the lysis of the RBCs before the WBCs. The WBCs are then pelleted by centrifugation, leaving the empty RBC membranes (ghosts) and hemoglobin, respectively, in suspension and solution.
Tissue Samples
Fresh or frozen tissue samples must be dissociated before DNA isolation procedures can be started. Grinding the frozen tissue in liquid nitrogen, homogenizing the tissue, or simply mincing the tissue using a scalpel can disrupt whole tissue samples. Fixed embedded tissue has to be deparaffinized by soaking in xylene (a mixture of three isomers of dimethylbenzene). Less toxic xylene substitutes, such as Histosolve, Anatech Pro-Par, or ParaClear, are also often used for this purpose. After xylene treatment, the tissue is usually rehydrated by soaking it in decreasing concentrations of ethanol.
Microorganisms
Some bacteria and fungi have tough cell walls that must be broken to allow the release of nucleic acid. Several enzyme products, e.g., lyzozyme or zymolyase, that digest cell wall polymers are commercially available. Alternatively, cell walls can be broken mechanically by grinding or by vigorously mixing with glass beads. Gentler enzymatic methods are less likely to damage chromosomal DNA and thus are preferred for methods involving larger chromosomal targets as opposed to plasmid DNA.
Treatment with detergent (1% sodium dodecyl sulfate) and strong base (0.2 M NaOH) in the presence of Tris base, ethylenediaminetetraacetic acid (EDTA), and glucose can also break bacterial cell walls.
Boiling in 8% sucrose, 8% Triton X-100 detergent, Tris buffer, and EDTA after lysozyme treatment releases DNA that can be immediately precipitated with alcohol. DNA extracted with NaOH or boiling procedures is denatured (single-stranded) and may not be suitable for methods such as restriction enzyme analysis that require double-stranded DNA. The advantage of these types of
extraction is their speed and simplicity. Amplification methods will work with this type of DNA isolation.
Organic Isolation Methods
After release of DNA from the cell, further purification requires removal of contaminating proteins, lipids, carbohydrates, and cell debris. This is accomplished using a combination of high salt, low pH, and an organic mixture of phenol and chloroform. The combination readily dissolves hydrophobic contaminants such as lipids and lipoproteins, collects cell debris, and strips away most DNA associated proteins. Isolation of small amounts of DNA from challenging samples such as fungi
can be facilitated by pretreatment with cetyltrimethylammonium bromide, a cationic detergent that efficiently separates DNA from polysaccharide contamination. To avoid RNA contamination, RNAse, an enzyme that degrades RNA, can be added at this point. Alternatively, RNAse may also be added to the resuspended DNA at the end of the procedure.
When phenol and chloroform are added to the hydrophilic cleared cell lysate, a biphasic emulsion forms. Centrifugation will settle the hydrophobic layer on the bottom, with the hydrophilic layer on top. Lipids and other hydrophobic components will dissolve in the lower hydrophobic phase. DNA will dissolve in the upper aqueous phase. Amphiphilic components, which have both hydrophobic and hydrophilic properties as well as cell debris, will collect as a white precipitate at the interface between the two layers.
The upper phase containing the DNA is collected, and the DNA is then precipitated using ethanol or isopropanol in a high concentration of salt (ammonium,potassium or sodium acetate, or lithium or sodium chloride). The ethyl or isopropyl alcohol is added to the upper phase solution at 2:1 or 1:1 ratios, respectively, and the DNA forms a solid precipitate.
The DNA precipitate is collected by centrifugation. Excess salt is removed by rinsing the pellet in
70% ethanol, centrifuging and discarding the ethanol supernatant, and then dissolving the DNA pellet in rehydration buffer, usually 10 mM Tris, 1 mM EDTA (TE), or water.
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| General scheme of Organic DNA isolation |
Inorganic Isolation Methods
Safety concerns in the clinical laboratory make the use of caustic reagents such as phenol undesirable. Methods of DNA isolation that do not require phenol extraction have, therefore, been developed and are used in many laboratories. Initially, these methods did not provide the efficient
recovery of clean DNA achieved with phenol extraction; however, newer methods have proven to produce high-quality DNA preparations in good yields.
Inorganic DNA extraction is sometimes called “salting out”. It makes use of low pH and high salt conditions to selectively precipitate proteins, leaving the DNA in solution. The DNA can then be precipitated as described above using isopropanol pelleted and resuspended in TE buffer or water.
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| General scheme of Organic DNA isolation |
Solid-Phase Isolation
More rapid and comparably effective DNA extraction can be performed using solid matrices to bind and wash the DNA. Silica-based products were shown to effectively bind DNA in high salt conditions. Many variations on this procedure have been developed, including use of diatomaceous earth as a source of silica particles. More modern systems can be purchased with solid matrices in
the form of columns or beads. Columns come in various sizes, depending on the amount of DNA to be isolated. Columns used in the clinical laboratory are usually small “spin columns” that fit inside microcentrifuge tubes. These columns are commonly used to isolate viral and bacterial DNA from serum, plasma, or cerebrospinal fluid. They are also used routinely for isolation of cellular DNA in genetics and oncology. Preparation of samples for isolation of DNA on solid-phase media starts with cell lysis and release of nucleic acids, similar to organic and inorganic procedures. Specific buffers are used to lyse bacterial, fungal, or animal cells. Buffer systems designed for specific applications (e.g., bacterial cell lysis or human cell lysis) are commercially available.
For solid-phase separation, the cell lysate is applied to a column in high salt buffer, and the DNA in solution adsorbs to the solid matrix. After the immobilized DNA is washed with buffer, the DNA is eluted in a specific volume of water, TE, or other low salt buffer. The washing solutions and the eluant can be drawn through the column by gravity, vacuum, or centrifugal force. DNA absorbed
to magnetic beads is washed by suspension of the beads in buffer and collection of the beads using a magnet applied to the outside of the tube while the buffer is aspirated or poured off. The DNA IQ system (Promega) uses a magnetic resin that holds a specific amount of DNA (100 ng). When the DNA is eluted in 100µL, the DNA concentration is known, 1 ng/µL, and ready for analysis.
Solid-phase isolation is the methodology employed for several robotic DNA isolation systems such as Roche MagnaPure and Qiagen BioRobot, which use magnetized glass beads or membranes to bind DNA. These systems are finding increased use in clinical laboratories for automated isolation of DNA from blood, tissue, bone marrow, plasma, and other body fluids. A measured amount of sample, e.g., 200–400µL of whole blood or 10–50 mg of tissue, in sample tubes is placed into the instrument along with cartridges or racks of tubes containing the reagents used for isolation. Reagents are formulated in sets depending on the type and amount of starting material. The instrument is then programmed to lyse the cells and isolate and elute the DNA automatically.
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| Isolation of DNA on solid media
Crude Lysis
Although high-quality DNA preparations are tantamount to successful procedures, there are circumstances that either preclude or prohibit extensive DNA purification. These include screening large numbers of samples by simple methods (e.g., electrophoresis with or without restriction enzyme digestion and some amplification procedures), isolation of DNA from limited amounts of starting material, and isolation of DNA from challenging samples such as fixed, paraffin-embedded tissues. In these cases, simple lysis of cellular material in the sample will yield sufficiently useful DNA for amplification procedures.
Protelytic Lysis of Fixed Material
Simple screening methods are mostly used for research purposes in which large numbers of samples must be processed. This is usually not done in the clinical laboratory. In contrast, the analysis of paraffin samples is frequently performed in the clinical laboratory. Fixed tissue is more conveniently accessed in the laboratory and may sometimes be the only source of patient material. Thin sections are usually used for analysis, although sectioning is not necessary with very small samples such as needle biopsies. Paraffin-embedded specimens must be dewaxed with xylene or other agents and then rehydrated before nucleic acid isolation. For some tests, such as somatic mutation analyses, a separate stained serial section can be examined microscopically to identify tumor cells. The identifiable areas of tumor can then be isolated directly from the slide by simple scraping in buffer (microdissection) or laser capture and deposited into microcentrifuge tubes.
Before lysis, cells may be washed by suspension and centrifugation in saline or other isotonic buffer. Reagents used for cell lysis depend on the subsequent use of the DNA. For simple screens, cells can be lysed in detergents such as SDS or Triton. For use in PCR amplification, cells may be lysed in a mixture of Tris buffer and proteinase K. The proteinase K will digest proteins in the sample, lysing the cells and inactivating other enzymes. The released DNA can be used directly in the amplification reaction.
Extraction with chelating resin
Chelex is a cation-chelating resin that can be used for simple extraction of DNA. A suspension of 10% chelex resin beads is mixed with specimen, and the cells are lyzed by boiling. After centrifugation of the suspension, DNA in the supernatant is cooled and may be further extracted with chloroform before use in amplification procedures. This method is most commonly used in forensic applications but may also be useful for purification of DNA from clinical samples and fixed, paraffin-embedded specimens.
Other rapid extraction methods
With the advent of PCR, new and faster methods for DNA isolation have been developed. The minimal sample requirements of amplification procedures allow for the use of material previously not utilizable for analysis. Rapid lysis methods (produced by Sigma or Epicentre Technologies) and DNA extraction/storage cards (produced by Whatman) provide sufficiently clean DNA that
can be used for amplification.
Isolation of Mitochondrial DNA
There are two approaches to the isolation of mitochon
drial DNA from eukaryotic cells. One method is to first
isolate the mitochondria by centrifugation. After cell
preparations are homogenized by grinding on ice, the
homogenate is centrifuged at low speed (700–2600 ϫ g)
to pellet intact cells, nuclei, and cell debris. The mito
chondria can be pelleted from the supernatant in a second
high-speed centrifugation (10,000–16,000 ϫ g). The
mitochondria can be lysed with detergent and the lysate
treated with proteinase to remove protein contaminants.
Mitochondrial DNA can then be precipitated with cold
ethanol and resuspended in water or appropriate buffers for analysis. The second approach to mitochondrial DNA preparation is to isolate total DNA as described above. The
preparation will contain mitochondrial DNA that can be analyzed within the total DNA background by hybridization or PCR.
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