November 1, 2005 (Vol. 25, No. 19)
Nucleic Acid Sample Preparation Using the ChargeSwitch Technology
Since the invention of PCR in 1983, molecular biology has accelerated biomedical research efforts and enabled major scientific breakthroughs at an unprecedented rate. PCR-related methods have subsequently become diversified from their original conception as simple amplification tools of DNA fragments to quantitative PCR (qPCR) of both DNA and RNA.
Simultaneously, nucleic acid purification techniques have also impacted the molecular biology revolution. Without methods that enable the purification of DNA and RNA, fundamental molecular biology methods including cloning, gene expression analysis, and DNA sequencing would not be possible. A new technology is now simplifying nucleic acid purification and allowing for ever more sophisticated molecular biology applications.
During the 1980s, salting out and organic solvent-based approaches were typically used to purify nucleic acids. From the early 1990s, silica-based formats, ranging from solutions to membranes, have been utilized. Although any of these methods are suitable for isolation of DNA and RNA for most applications, they can leave behind contaminating organic solvents, chaotropic salts, or ethanol that can be inhibitory in downstream applications. Therefore, additional considerations of nucleic acid purification technology must be addressed when purifying nucleic acids for some of today’s more demanding applications.
Factors necessary to consider when evaluating nucleic acid purification techniques include DNA and RNA yield and purity, the labor-intensiveness of the procedure, the potential handling and disposal of toxic materials, and carryover of organic species from the purification process into the final DNA or RNA sample, which can interfere with subsequent analysis.
Keeping Pace with the Marketplace
Until the late 1990s, little progress had been made in new developments of nucleic acid purification to keep pace with the unprecedented range of demanding applications of the life science marketplace. Then, in 2002, DNA Research Innovations (DRI; Kent, U.K.) developed a surface coating with a charge dependent on the pH of the surrounding biological solution, and called the technology ChargeSwitch. (DRI is now a wholly owned subsidiary of Invitrogen in Carlsbad, CA.)
Invitrogen’s ChargeSwitch Technology uses a switchable surface charge dependent on the pH of the surrounding buffer to purify nucleic acids. In conditions of pH <6.5, the ChargeSwitch surface has a positive charge that selectively binds the negatively charged nucleic acid backbone. Proteins and other contaminants are removed in an aqueous wash buffer.
For elution, the charge on the ChargeSwitch surface is switched off by raising the pH to 8.5. Purified nucleic acid elutes instantly and is ready for downstream use. The ChargeSwitch Technology method uses low-salt, water-based buffers and avoids the introduction of organic solvents, ethanol, or concentrated chao-tropic salts.
The technology is particularly useful for purification of nucleic acids from limited samples, yields nucleic acids without carryover of substances which interfere with downstream applications, and is ideal for multiple applications, including but not limited to forensics, sensitive PCR reactions, and high throughput experiments.
Forensics laboratories routinely process crime scene samples for DNA analysis. The aim is to generate STR (short tandem repeat) profiles for human identification that meet the stringent requirements of the criminal justice system. Conventional DNA purification methods involve salting out with harsh organic solvents, anion exchange, and silica-based extraction, which can compromise subsequent DNA processing in STR profiling.
Additionally, crime scene samples vary in quantity and quality, adding to the challenge of reliably purifying DNA suitable for forensics. By eliminating chemicals that can interfere with the integrity of DNA samples, ChargeSwitch Technology can offer sensitivity for purifying and quantitating DNA from forensic samples, thereby meeting the criminal justice system requirements for STR profiling.
ChargeSwitch Technology produces nucleic acids that can be used in sensitive PCR reactions, such as the detection of low copy number genes for either real-time quantitative PCR or traditional PCR. The most common RNA or DNA purification techniques use reagents that can inhibit thermostable polymerase activity (PCR reactions), even though those reagents are difficult to detect when measuring nucleic acid quality on gels or by UV spectrophotometry.
Data shows that even low levels of alcohols or chaotropic salts can completely inhibit polymerase activity, thereby causing PCR to fail. The ChargeSwitch Technology was designed to use 100% aqueous solutions. Ethanol, phenol, chloroform, and ionic chaotropes have all been completely eliminated, removing any possibility of PCR failure due to their presence.
More and more laboratories are investing in techniques requiring the high throughput purification of nucleic acids and demanding faster purification methods. Traditional nucleic acid purification technologies are readily integrated into robotic system, but they require time-consuming centrifugation or vacuum steps, which can lead to bottlenecks.
By avoiding the use of these steps, ChargeSwitch Technology accelerates the isolation of plasmids, RNA, PCR products, or genomic DNA, allowing scientists to move on to the next experimental steps more quickly. Additionally, ChargeSwitch Technology surface chemistry can be coated onto any surface, making possible flexible new formats for these streamlined processes.
As novel applications requiring ultra-pure DNA and RNA preparations have been developed, researchers have sought improved nucleic acid purification technologies that can deliver inhibitor-free DNA and RNA. ChargeSwitch Technology was developed to meet the needs of life science innovators. With its simplicity and aqueous-based chemistry, ChargeSwitch Technology can meet the current needs of scientists performing demanding molecular biology applications and will allow the development of the next generation of molecular biology applications.