DNA precipitation is a fundamental technique in molecular biology used to isolate and concentrate DNA from an aqueous solution. This process is necessary after DNA has been extracted from a sample, which often leaves the nucleic acid dispersed in a dilute liquid containing various contaminants. Converting the dissolved DNA into a compact, solid form allows researchers to easily separate it from the remaining liquid components. This purification and concentration step prepares the genetic material for downstream analysis.
The Role of DNA Precipitation
Scientists precipitate DNA to significantly increase the concentration of a sample or remove residual contaminants that interfere with later experiments. This technique is often the final purification step in traditional DNA extraction protocols, yielding a highly concentrated and clean sample suitable for applications requiring a specific minimum amount of genetic material.
The purified product is routinely used to prepare samples for next-generation sequencing, which requires DNA to be highly concentrated and free of salts. In forensic science, precipitation helps concentrate minute traces of DNA from crime scene evidence for genetic testing. It is also useful in academic research for preparing DNA for cloning, enzyme digestion, or polymerase chain reaction (PCR).
Required Reagents and Materials
The process relies on the precise combination of a salt solution and an alcohol to force the DNA out of the solution. The DNA sample is initially dissolved in a buffer, typically water or a low-salt solution.
A high-concentration salt solution, such as 3 M Sodium Acetate at pH 5.2, is added first, typically at one-tenth the volume of the DNA sample. The salt provides positively charged ions, like sodium (Na+), which interact with the negatively charged DNA molecule. Cold alcohol is then introduced; 100% Ethanol or Isopropanol are the preferred choices. Ethanol requires approximately two to two-and-a-half times the volume of the original DNA solution, while Isopropanol requires only about 0.7 to 1 volume.
Step-by-Step DNA Precipitation
The procedure begins by ensuring the DNA is in an appropriate liquid volume that can be handled in a centrifuge tube. The first chemical step involves adding the salt solution, such as 3 M Sodium Acetate, directly to the DNA sample and mixing thoroughly.
Next, the cold alcohol is added to the mixture, which must be gently inverted several times to ensure full mixing without causing shearing damage to the DNA. Using cold alcohol, often stored at -20°C, and chilling the sample promotes the clumping of DNA into a visible, solid form. Incubating the mixture at -20°C for at least one hour, or even overnight, is recommended, especially for samples with low DNA concentrations.
Following incubation, the mixture is subjected to high-speed centrifugation, typically at 12,000 x g or higher for 15 to 20 minutes. This spinning forces the solidified DNA to the bottom of the tube, forming a small, concentrated mass known as a pellet. The liquid portion, called the supernatant, is then carefully removed, leaving only the DNA pellet behind.
The Underlying Chemical Mechanism
The success of DNA precipitation is rooted in the unique structure of the DNA molecule and its interaction with the surrounding solvent. The DNA backbone is highly negatively charged due to the phosphate groups. In an aqueous solution, water molecules surround the DNA, forming a hydration shell that keeps the DNA soluble and prevents repulsion between the negative charges.
The added salt, such as sodium acetate, introduces a high concentration of positively charged ions, like Na+, into the solution. These positive ions neutralize the negative charges on the phosphate backbone, shielding the repulsion between strands. However, the high dielectric constant of water weakens the electrostatic attraction, meaning the DNA remains dissolved.
Introducing alcohol, which has a much lower dielectric constant than water, forces the DNA out of solution. The alcohol significantly reduces the ability of the solution to screen the charges, allowing the positive salt ions to form stable ionic bonds with the negative phosphate groups. This neutralization removes the hydrophilic nature of the DNA, making it insoluble and causing it to aggregate and precipitate as a solid mass.
Post-Precipitation Handling and Storage
Once the DNA pellet is separated from the supernatant, it is crucial to wash it to remove residual salts and contaminants that co-precipitated with the DNA. This is accomplished by adding a solution of 70% ethanol, which dissolves the salts but not the precipitated DNA. The tube is gently inverted to wash the pellet, followed by a second, shorter centrifugation step to re-pellet the clean DNA.
After the wash, the 70% ethanol is removed, and the DNA pellet is allowed to air-dry, usually for 5 to 15 minutes, until the last traces of alcohol have evaporated. It is important to avoid over-drying the pellet, as this makes it difficult to re-dissolve the DNA in the final buffer. The final step is to re-suspend the clean, dry DNA in a small volume of a suitable buffer, such as sterile water or TE buffer, for stability. The re-suspended DNA can then be stored at -20°C for long-term preservation.