DNA purification separates deoxyribonucleic acid (DNA) from other cellular components and contaminants after initial extraction. This step ensures isolated DNA is clean and ready for various downstream applications. It primarily involves removing non-DNA molecules like proteins, RNA, and lipids, often co-extracted from biological samples. The goal is a highly pure DNA sample, free from anything that might interfere with subsequent experiments.
Why DNA Purification is Essential
DNA extraction often yields a mixture of DNA and cellular debris. Common contaminants include proteins (which inhibit enzyme activity), RNA (which can be mistaken for DNA), and lipids and carbohydrates (which interfere with clarity and efficiency). Residual salts and organic solvents (e.g., phenol, chloroform) can also remain. These impurities compromise accuracy and success, leading to inaccurate results, failed reactions, or reduced sensitivity. Purifying DNA is necessary for reliable, reproducible outcomes in research and diagnostic settings.
Common Purification Techniques
Several methods purify DNA, each relying on different chemical and physical principles. These techniques aim for high purity and yield, varying in complexity, speed, and suitability for different sample types and applications.
Alcohol precipitation, using ethanol or isopropanol, is a widely used method that leverages DNA’s insolubility in high concentrations of these alcohols and salts. A common procedure involves adding a salt (e.g., sodium acetate) to the DNA solution, then cold ethanol or isopropanol. The salt neutralizes DNA’s negative charges, causing it to aggregate and precipitate into a visible pellet. The pellet is collected by centrifugation, washed with 70% ethanol to remove residual salts, air-dried, and re-dissolved in buffer. Isopropanol requires less volume and can be performed at room temperature, making it suitable for larger samples.
Silica-based column purification, often found in commercial kits, is another common approach. It relies on DNA selectively binding to a silica membrane in high concentrations of chaotropic salts. These salts disrupt water’s hydrogen bonds, making DNA adhere strongly to silica. The DNA-bound column is washed with alcohol-based buffers to remove non-binding contaminants like proteins and polysaccharides. Finally, purified DNA is eluted from the membrane using a low-salt buffer or nuclease-free water. This technique is fast, easy to use, and yields high-quality DNA, making it popular.
Magnetic bead-based purification is a flexible, often automatable alternative. It uses microscopic magnetic beads coated to bind DNA under specific buffer conditions. Once DNA binds, an external magnetic field draws the beads (and bound DNA) to the vessel side, allowing easy removal of the contaminant-containing supernatant. Multiple wash steps are performed while beads are immobilized by the magnet to ensure thorough impurity removal. Purified DNA is then released from the beads by adding an elution buffer and removing the magnetic field; this technique minimizes manual handling and centrifugation, making it efficient for high-throughput applications and reducing physical stress on DNA.
Assessing DNA Purity and Quantity
After purification, confirming DNA quality and quantity is essential before downstream applications. Researchers commonly use spectrophotometry and gel electrophoresis.
Spectrophotometry measures light absorbed by DNA at specific wavelengths. DNA absorbs UV light most strongly at 260 nm, allowing concentration calculation. Purity is assessed by absorbance ratios. The A260/A280 ratio indicates protein contamination; a ratio of approximately 1.8 is considered pure. A lower ratio suggests protein, while a higher ratio can indicate RNA. The A260/A230 ratio indicates contamination by salts, organic solvents, or carbohydrates; a ratio between 2.0 and 2.2 is considered pure. Low A260/A230 ratios suggest these contaminants.
Gel electrophoresis visually assesses DNA integrity and detects contaminants like RNA or degraded DNA. DNA samples are loaded into an agarose gel, where an electric current causes fragments to migrate based on size and charge. Intact, high molecular weight DNA appears as a distinct, compact band. A smear below the main DNA band often indicates degradation or shearing. RNA contamination can appear as fast-migrating smears or distinct bands at the bottom of the gel, separate from larger DNA; this visual inspection complements spectrophotometric readings, providing a comprehensive picture of DNA quality.
Applications of Purified DNA
Purified DNA is a prerequisite for many molecular biology techniques and research applications. Its cleanliness ensures reliability and success in downstream processes.
Purified DNA serves as a template for Polymerase Chain Reaction (PCR), amplifying specific sequences. DNA sequencing, which determines nucleotide order, also requires highly pure DNA for accurate results. It is also essential for cloning, where a gene is inserted into a vector for replication or expression. Restriction enzyme digestion, cutting DNA at specific sites, also requires pure DNA for optimal enzyme function without contaminant inhibition. Advanced applications like microarray analysis and diagnostic tests rely on purified DNA quality for sensitivity and specificity.