James Netterwald, PhD, is a freelance science and medical writer based in New Jersey. His writing serves every life science industry.
DNA purification is the gateway step to molecular biology and genomics applications ranging from cloning and PCR to genome sequencing and microarray analysis. But choosing the best purification kit to fit your favorite application can be tricky. For example, if you need to isolate genomic DNA, you definitely want a kit that allows for a gentle treatment of that type of nucleic acid, and one that is not meant to isolate plasmid DNA. Here we highlight some popular options.
Salt-precipitation methods
The MasterPure™ Complete DNA Purification Kit from Epicentre, an Illumina company, uses a proprietary, gentle salt-precipitation method. With a diverse sample range that includes blood, fixed tissue, plants, yeast, insects and more, these kits deliver high yields of nucleic acids, even from small samples, with minimal loss in less than 30 minutes.
“With these kits, the user eliminates the need for additional cleanup steps before sample preparation for sequencing or other molecular-biology applications,” says Cristine Kinross, senior product manager at Epicentre. The kit’s chemistry is designed to remove proteins and other cellular debris without toxic chemicals that must later be removed, thereby improving yields by avoiding washes that result in loss of nucleic acid.
“The exceptional purity of nucleic acids recovered using MasterPure enables samples to go directly into sequencing library-preparation applications,” Kinross says, though other applications, including microarrays, qPCR and cloning are also compatible.
Multiple chemistries
Promega offers an array of nucleic acid-purification kits in its portfolio, employing several different strategies. The company’s Wizard® Genomic DNA Purification Kit, for instance, works by differential precipitation, and its ReliaPrep™ kits (eg, the ReliaPrep 96 gDNA Miniprep HT System) uses a silica gel in a microcentrifuge column. Promega also has kits based on magnetic particle-based purification.
“While exact compositions of solutions may vary, the basic process of lyse, bind, wash and elute is used with different binding chemistries, such as silica or cellulose, and different purification formats, such as columns or magnetic particles, in manual or automated purification protocols,” says Eric Vincent, global product manager for genomics products at Promega. For some very important sample types (e.g., formalin-fixed paraffin-embedded (FFPE) samples), more specialized protocols or treatments are required.
“We have developed unique binding/wash buffers that effectively remove substances that are inhibitory to downstream applications, optimized systems to allow researchers to capture small amounts of nucleic acid and elute in small volumes (< 30 μl) and have developed automated solutions for a variety of chemistries and throughputs,” says Vincent.
These kits provide complete solutions that enable elution of nucleic acid in very small volumes while providing washing conditions that eliminate carryover of inhibitors into the eluate. These kits enable the purification of nucleic acids for PCR, Sanger sequencing, next-generation sequencing, microarrays and more.
Basic silica binding
Qiagen’s nucleic acid-purification kits rely on the binding of DNA to a silica matrix, which is attached to a column. Inhibitors are washed away, and pure, intact DNA subsequently is eluted from the column at high yields, leading to higher-quality next-generation DNA sequencing libraries.
“The high yield becomes even more crucial when dealing with very small sample sizes or difficult samples like FFPE slides,” says Marco Polidori, associate global product manager for sample technologies at Qiagen. FFPE-generated DNA samples can contain “random cytosine deamination” events, which appear as artifactual single nucleotide polymorphisms. But Qiagen’s GeneRead DNA FFPE Kit, for instance, removes deaminated cytosines before they can lead to erroneous results in NGS experiments.
“Most of our kits are already proven in NGS applications, from whole-genome sequencing of free-circulating tumor DNA to microbiomes,” says Polidori. These kits enable the user to obtain high-quality DNA from a large variety of samples proven to work with next-generation sequencing, remove C-to-T artifacts in FFPE samples, deliver genomic DNA of high molecular weight (larger than 200 kb with the MagAttract HMW DNA kit) in a very short amount of time with either manual or automated procedures and more.
Chaotropic salts
Sigma-Aldrich offers the GenElute™ Mammalian Genomic DNA Purification Kit, which is based on chaotropic-salt chemistry. In a single chaotropic-salt-containing solution, samples are lysed to ensure the thorough denaturation of macromolecules. Adding ethanol causes DNA to bind to a silica membrane when spun in a microcentrifuge tube. Contaminants are washed away, and DNA is eluted in Tris buffer.
With a silica specially selected to isolate genomic DNA, the kit enables users to purify high-quality DNA from a variety of mammalian sources, including cultured cells, tissues (including rodent tails), and fresh whole blood or white blood cells.
“The kit combines the advantages of silica binding with a microspin format and eliminates the need for expensive resins, alcohol precipitation and hazardous organic compounds such as phenol and chloroform,” according to the company website. More importantly, genomic DNA purified by this kit can be applied to restriction endonuclease digestions, PCR, Southern blots, sequencing reactions and more.
Correction (19 December 2013): This article has been updated to correct the name of a Qiagen kit.