Polymerase Chain Reaction (PCR) is a widely used molecular biology technique that amplifies specific DNA sequences, allowing for detailed analysis even from minute starting quantities. The reliability and accuracy of PCR experiments depend heavily on the purity of all reagents, with water being a fundamental component of the reaction mixture. PCR grade water is a highly purified form of water, specifically treated to be free of contaminants that could interfere with the sensitive enzymatic reactions central to PCR. Its exceptional purity helps ensure consistent and reproducible results in various molecular biology applications.
Understanding Water Contaminants
Regular laboratory water contains various impurities that can significantly impede PCR amplification, leading to failed reactions, reduced efficiency, or inaccurate results. Understanding these interferences is essential for producing water suitable for sensitive molecular applications.
Nucleases, specifically deoxyribonucleases (DNases) and ribonucleases (RNases), pose a significant threat to PCR. These enzymes degrade DNA and RNA templates, as well as primers. Even minute amounts of these enzymes can compromise nucleic acid integrity, leading to a lack of amplification or false negative outcomes.
PCR inhibitors represent a diverse group of substances that can directly interfere with the reaction process. Metal ions, such as zinc, tin, iron, and copper, can inhibit DNA polymerase activity. Organic compounds like humic acids and phenol, often found in environmental samples, can also bind to DNA or inhibit polymerase enzymes. Additionally, detergents and high concentrations of salts can disrupt the reaction buffer or enzyme function, reducing PCR efficiency.
Microorganisms and their byproducts, such as endotoxins, can also be present in water. While filtration typically removes the organisms themselves, their cellular components can still interfere with PCR. High concentrations of ionic impurities can disrupt the precise ionic balance required for optimal polymerase activity and primer binding within the PCR buffer.
Methods for Purification
Producing water suitable for PCR requires a combination of advanced purification techniques, as no single method can remove all types of contaminants. Multi-stage purification systems are commonly employed to achieve the necessary high level of purity. This comprehensive approach ensures the removal of ions, organic molecules, particulates, nucleases, and microorganisms.
Distillation is one of the oldest purification methods, involving boiling water and then condensing the steam. This process effectively removes non-volatile impurities, dissolved solids, and some microorganisms. However, volatile organic compounds can carry over, and distillation is energy-intensive and slow, making it less efficient for large-scale, high-purity water production alone.
Deionization (DI) utilizes ion-exchange resins to remove dissolved inorganic salts and minerals. Water passes through resin beds where undesirable ions are exchanged for hydrogen (H+) and hydroxyl (OH-) ions, which then combine to form water molecules. While highly effective at removing ions, deionization does not remove organic compounds, particles, or microorganisms. Mixed-bed deionizers, containing both cation and anion exchange resins, can achieve very high resistivity values, indicating low ionic content.
Reverse osmosis (RO) is a membrane-based technology that forces water through a semi-permeable membrane under pressure. This membrane has pores small enough to reject most dissolved salts, organic molecules, particles, bacteria, and viruses, including pyrogens. RO is a highly effective pre-treatment step, removing a broad spectrum of contaminants and reducing the load on subsequent purification stages. It typically removes 90% to 99% of contaminants.
Ultraviolet (UV) irradiation is used to inactivate microorganisms and oxidize organic compounds. UV lamps, typically emitting at 254 nm, damage the DNA of microbes, preventing their replication. A second UV wavelength, usually 185 nm, can be used to break down trace organic molecules into charged ions, which can then be removed by subsequent deionization steps.
Sub-micron filtration involves passing water through filters with very small pore sizes, commonly 0.22 µm or 0.1 µm. These filters physically remove particulate matter and bacteria. For molecular biology applications, ultrafiltration (UF) polishers, often with a nominal molecular weight cut-off around 13,000 Daltons, are used as a final step to remove nucleases and other large biomolecules. This ensures the water is free from enzymes that could degrade nucleic acids.
Ensuring Quality and Proper Storage
Maintaining the purity of PCR grade water after production is as important as the purification process itself. Regular quality control checks and diligent storage practices are necessary to prevent re-contamination and ensure optimal performance in sensitive applications.
Water purity is assessed using several quality control measures. Conductivity or resistivity measurements indicate the level of ionic impurities present in the water. Ultrapure water, suitable for PCR, typically achieves a resistivity of 18.2 MΩ·cm at 25°C, signifying extremely low ion concentrations and confirming deionization effectiveness.
Functional tests are also performed to confirm the absence of specific PCR inhibitors and nucleases. Nuclease assays involve incubating the water with a known DNA or RNA substrate and then checking for degradation using gel electrophoresis. PCR inhibition tests assess if the water allows for efficient amplification of a target DNA sequence, ensuring no inhibitory substances are present. Testing for microbial contamination also confirms sterilization effectiveness.
Proper storage is essential to prevent re-contamination of purified water. PCR grade water should always be stored in sterile, nuclease-free containers, preferably made of polypropylene, which are less likely to leach contaminants. Aliquoting the water into smaller volumes upon opening can minimize the risk of widespread contamination from repeated access.
To further maintain purity, avoid direct contact between the water and non-sterile surfaces or instruments. Using dedicated pipettes and filter tips for handling PCR reagents, including water, helps prevent aerosol and cross-contamination. Storing PCR grade water and other reagents in a dedicated area, separate from where amplified DNA products are handled, reduces the chance of carryover contamination. While autoclaving can help maintain sterility, it does not remove all chemical inhibitors or nucleases. For optimal performance, use freshly prepared or recently opened PCR grade water, as purity can degrade over time, even with proper storage at recommended temperatures.