Silica-based spin columns have long been used as an alternative to traditional phenol chloroform extraction for purifying DNA and RNA. However, a major disadvantage of these is that they involve multiple, tedious wash and spin steps, adding time to protocols and greatly increasing the risk of operator error. Silica-based purification methods are also highly susceptible to contamination with buffer components and sheared nucleic acid impurities, leading to numerous problems downstream, and they generate significant quantities of waste. Designed to overcome these issues, GenElute™-E Single Spin DNA and RNA purification kits from Merck KGaA, Darmstadt, Germany, are a disruptive technology that promises to replace silica-based methods. Here, we explain how the simplified workflow and superior performance of GenElute™-E technology yields more robust results.
Silica-based methods are laborious and prone to contamination
Silica-based extraction of DNA or RNA from samples including cells, tissues, or blood follows a bind-wash-elute protocol. First, nucleic acids are captured on a silica membrane column in the presence of a chaotropic salt such as guanidine hydrochloride, then an ethanol-based salt solution is used to wash away other biomolecules. The nucleic acids are then eluted with Tris buffer or water.
Although relatively straightforward, a drawback of this method is that it involves considerable hands-on manipulation. Moreover, chaotropic salts can falsely elevate nucleic acid concentration estimates and interfere with downstream enzymatic processes such as qPCR. A further issue arises from the necessity for multiple spin steps since these can shear nucleic acids to reduce the amount of full-length product available for analysis. With high molecular weight genomic DNA being critical for applications such as next-generation sequencing (NGS), shearing should be avoided.
GenElute™-E improves nucleic acid purification
Instead of relying on multiple wash and spin steps, GenElute™-E Single Spin DNA and RNA purification kits use negative chromatography to isolate nucleic acids. Based on a novel lysis method, sample specific enzymes optimize the procedure and eliminate the need for an overnight step, with size exclusion employed to allow large DNA and RNA nucleic acid molecules to flow through the column while smaller protein, lipid, and ionic components are retained. The advantages of this approach compared to silica-based nucleic acid extraction include a simplified workflow, fewer impurities, and reduced waste, all of which contribute to higher quality nucleic acid purification.
Simplified protocol
A primary advantage of GenElute™-E Single Spin DNA and RNA purification kits is that they enable researchers to purify nucleic acids in a fraction of the time required for silica bind-wash-elute procedures. Just a simple spin and 3 minutes hands-on manipulation are required to purify DNA or RNA using GenElute™-E purification kits compared to multiple centrifugation steps and 45 minutes hands-on time using silica-based methods. Moreover, with GenElute™-E Single Spin technology, the entire purification process can be completed in as little as 15 minutes, whereas a minimum of 45 minutes is necessary for silica-based methods.
Fewer impurities
One of the most common problems associated with silica-based DNA and RNA purification methods is that they invariably introduce denaturing salts and organic solvents (e.g. ethanol) to the sample. These contaminants can be carried over into downstream applications such as qPCR, where they can inhibit enzymatic reactions to decrease assay sensitivity. They can also affect the ultraviolet (UV) measurements used in nucleic acid quantitation. For example, Tris, EDTA, and guanidinium all absorb strongly at 230 nm and can bleed into the 260 nm absorbance range used to calculate DNA and RNA concentration, falsely elevating estimated concentrations and yield.
Compared to silica-based methods, GenElute™-E purification produces far fewer chemical contaminants (as evidenced by UV spectrophotometry and qPCR), translating to more accurate quantitation. The single-spin process also reduces the presence of sheared nucleic acid impurities (as evidenced by gel electrophoresis) to provide researchers with greater quantities of full-length product.
Reduced waste
GenElute™-E Single Spin DNA and RNA purification kits are an eco-friendly alternative to silica-based nucleic acid purification. Not only are fewer plastic-based components packaged with each kit, but plastic use is also considerably reduced while executing protocols in the lab, affording a 55% reduction in plastic waste compared to a standard, off-the-shelf silica-based product. Furthermore, by removing the many binding and washing steps associated with silica-based procedures, GenElute™-E purification kits eliminate hazardous liquid waste, resulting in disposal cost savings and reduced environmental impact.
GenElute™-E Single Spin DNA and RNA purification kits can directly replace most silica-based nucleic acid preparation methods. Offering simpler DNA and RNA isolation, better nucleic acid quality with fewer impurities, and reduced plastic and hazardous waste disposal compared to established methods, this novel technology looks set to change workflows.
To learn more about GenElute™-E Single Spin DNA and RNA purification kits, visit SigmaAldrich.com/singlespin
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