The first step of most molecular pathology tests is isolation of DNA or RNA from a patient specimen, by either manual or automated methods. Nucleic acid purification begins with lysis of the cells in the sample. Cell lysis liberates cellular macromolecules including proteins, lipids, and nucleic acids. Cell lysis can be accomplished using a detergent solution to break cell membranes and remove lipids. Proteins are enzymatically degraded with protease, usually proteinase K, or selectively precipitated. Protein digestion is performed at about 56°C and will permanently denature many proteins but does not affect nucleic acids. This process is followed by selective extraction that takes advantage of the physical and chemical differences between nucleic acids and other cellular molecules, forming the basis for their isolation. The nucleic acid is then purified from the soluble contaminants produced in the extraction method by precipitation in an ethanol-salt solution. A variation on this theme that combines extraction and purification is the selective adsorption of nucleic acids to silica columns under chaotropic salt conditions. The isolated nucleic acid is then resuspended in a dilute salt buffer, for example, 10mM Tris/1 or 0.1mM EDTA pH 7.6 to 8.0 (TE buffer).
The initial lysis step is modified according to the specimen. If the specimen is fresh or frozen solid tissue, it is first homogenized in an appropriate buffer (often TE buffer). If the specimen is formalin-fixed, paraffin-embedded (FFPE) tissue, the paraffin is removed with an organic solvent such as xylenes, followed by rehydration through an alcohol series to a dilute salt buffer before protease digestion. FFPE tissue is also heated during the prolonged protease digestion step to reverse formalin cross-linking between proteins (primarily histones) and nucleic acids. Although this reduces the degree of nucleic acid shearing in subsequent vortexing or centrifugation steps, DNA longer than that packaged into a nucleosome (about 200 base pairs [bp]) is difficult to recover from FFPE tissue. Whole blood specimens may require a centrifugation step to remove erythrocytes prior to recovery of nucleic acids from leukocytes because of the inhibition of PCR by hemoglobin. This cell fractionation step generally is not performed in automated nucleic acid extraction instruments.
Organic (Phenol) Extraction
Nucleic acids have a strong negative charge because of the phosphate groups in the sugar-phosphate backbone, and thus are highly soluble in an aqueous environment. By contrast, proteins, lipids, and carbohydrates contain varying proportions of charged and uncharged domains producing hydrophobic and hydrophilic regions. This difference makes proteins entirely soluble in organic solutions or selective for the interface between the organic and aqueous phases during an organic extraction. This characteristic forms the basis for phenol: chloroform extraction, in which phenol is added to an aqueous solution containing cellular constituents, mixed, and then centrifuged to separate the aqueous and organic phases. If the pH of the extraction is near neutral, both DNA and RNA stay in the aqueous phase, while proteins are in the phenol: chloroform phase or aqueous-phenol interface. If the pH is acidic, the phosphate groups of DNA are preferentially neutralized, driving DNA into the organic phase (or interface) and allowing RNA to be selectively extracted. This method produces high-quality nucleic acids but is relatively labor-intensive, uses hazardous chemicals, and produces liquid organic waste.
Nucleic acids can be precipitated in an aqueous solution by the addition of concentrated ethanol and salt. Ethanol makes the solution hydrophobic, while salt increases the ionic strength of the solution, thereby reducing the repulsion of the negatively charged sugar-phosphate backbone of the nucleic acid. Centrifugation allows the precipitate to be collected and resuspended in a dilute salt buffer (TE buffer).
Chaotropic salts such as sodium iodide (NaI) or guani-dinium isothyocyanate (GITC) disrupt the structure of water, promoting the solubility of nonpolar substances, such as proteins, in polar solvents, such as water. Saturated chaotropic salts also promote the adsorption of nucleic acids to glass or silica columns. The nucleic acid is purified by a series of washing steps including reducing agents such as sodium azide to further remove contaminants and inhibit remaining enzymes. The nucleic acid is eluted from the column with a dilute, nonchaotropic salt buffer. Since the method is simple, fast, offered in commercial kits by several manufacturers, and adaptable to high-throughput robotic nucleic acid isolation, this method is widely used by clinical molecular laboratories.
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