by Caitlin Smith
The dawn of the kit era, when everything you needed for a routine yet important lab task could be conveniently purchased in a box, made life easier for anyone who works with genetic material. For example, kits for the extraction, isolation and preparation of a pure sample of genomic DNA (gDNA) are essential in many labs. “Highly pure DNA is essential for most sensitive downstream molecular biology applications, such as PCR, sequencing and cloning,” says Heather Secrist, chief executive officer of Genetic ID. “Therefore, choosing an appropriate genomic DNA extraction kit should first take into consideration the intended use of the DNA and then, ideally, also be easy to use, economical and rapid.” Looking for a genomic DNA purification kit can be overwhelming, because there are many out there to choose from. Different genomic DNA extraction kits yield different results, depending on the source of the gDNA—whether it’s cultured cells, whole blood, buffy coat, tissue, mouse tail, bacteria or formalin-fixed paraffin embedded (FFPE) samples. Here are some recent developments in genomic DNA preparation kits, and some factors to consider when choosing one.
The source of genomic DNA
A challenge for designers of gDNA preparation kits is to offer a system that can extract and purify DNA efficiently from a wide range of sources. For example, according to Nicole Neubauer, product manager at Promega, “the new generation of gDNA extraction kits [focuses] on high-quality DNA from tissue and blood samples. But other sample types, like mouse tail, buccal swab, FFPE tissue, plant, yeast or fungi, can be extracted with our gDNA product range, too.”
Similarly, Secrist says that Genetic ID’s Fast ID technology is designed to purify gDNA from a wide variety of sample types. “Because the Fast ID kit is suitable for such a broad array of sample matrices, including microbes, plant and animal tissue, clinical specimens, formalin-fixed paraffin embedded tissue and processed food,” says Secrist, “our customers range from testing laboratories to genetically modified crop technology providers, government laboratories and academic researchers.”
Life Technologies’ recent release, the MagMAX™ DNA Multi-Sample Kit, also can be used to prepare gDNA from many different types of samples, including blood, buffy coats, buccal swabs, cultured cells and a wide variety of tissues. “It utilizes magnetic particle technology, which offers a lot of advantages as it allows for scalability and [automation],” says Christopher Kim, product manager for DNA sample preparation and quantitation at Life Technologies. “And [the kit] has been validated with many of our Applied Biosystems kits for genetic analysis such as assays, [capillary electrophoresis] and next-generation sequencing.” Life Technologies also is preparing to launch its new MagMAX™ FFPE DNA Isolation Kit. “This kit follows the release of our MagMAX™ FFPE Total Nucleic Acid Isolation Kit,” says Kim. “Both kits are designed to require less hands-on time and less toxic reagents. From a technological perspective, the simplified process eliminates the arduous step of de-paraffination and allows for an automation-friendly workflow.”
QIAGEN offers a range of products to purify FFPE samples, one of the most challenging gDNA sources. The company’s recent release, the AllPrep DNA/RNA FFPE Kit, helps researchers purify gDNA and RNA. With other techniques, “If scientists want to isolate both genomic DNA as well as the RNA, [they have] to divide the biological sample into two and isolate both separately,” says Marc Egelhofer, manager for marketing public relations at QIAGEN, “which means more hands-on work and a higher amount of sample that needs to be consumed by the nucleic acid preparation.” QIAGEN’s AllPrep DNA/RNA FFPE Kit was designed to purify gDNA and total RNA simultaneously from FFPE tissue sections. “This means that pure DNA and RNA can be obtained from the entire sample, in contrast to other procedures where the biological sample is divided into two before being processed separately,” says Egelhofer. “This provides scientists with the maximum output with minimal sample consumption, while releasing the genomic DNA and the RNA without compromising integrity.” Another type of DNA and RNA co-purification kit is offered by Zymo Research; its ZR-Duet™ DNA/RNA MiniPrep kit can prepare DNA and RNA from the same sample, using cells or tissues.
Efforts to develop easier ways to extract gDNA with higher purity continue to raise the bar for genomic DNA preparation kits. Promega recently released its ReliaPrep™ Blood and Tissue gDNA Miniprep Systems for the high-yield extraction of gDNA (according to improved A260/A230 ratios) from both normal and particularly challenging samples. The kits are ready-to-use, and specially designed for small sample volumes. “For blood, purification [of four to 10 µg gDNA in as little as 50 µl elution] can be processed from as little as 200 µl whole blood, in less than one hour,” says Neubauer. “For larger blood samples, up to 10 ml of whole blood can be extracted. The system is fully scalable and can be automated. Protocols do not involve ethanol and can purify gDNA even from compromised samples, such as frozen blood.” The ReliaPrep™ gDNA Tissue Miniprep System purifies gDNA from up to 25 mg of tissue, also without alcohol washes, which can inhibit or interfere with downstream applications using the purified gDNA. “Researchers who tested our new kits use the extracted DNA for the most demanding applications, such as next-generation sequencing,” says Neubauer. Looking toward the future, she says, “additional improvements we are aiming for are fully scalable kits to enable researchers to reliably extract small and fragmented DNA samples that may also consist of FFPE starting material.”
G Biosciences offers kits specially designed to yield ultrapure gDNA, but they contain several extra steps to achieve the higher purity; its OmniPrep™ genomic DNA kits for ultrapure gDNA are fully scalable for larger-yield needs. The company also offers GET™ genomic DNA kits in a spin-column format, convenient for small samples. If you need gDNA that is protein-free, G Biosciences’ XIT™ genomic DNA kits use protein digestion and precipitation prior to gDNA purification. Purifying high molecular weight gDNA requires special conditions. These are found in G Biosciences’ MegaLong™ kit for isolating high molecular weight gDNA from a range of sample types.
Another challenge to achieving gDNA purity is fending off contamination in the final gDNA sample—and the source of this contamination can be surprising. “Most kits on the market do have traces of bacterial DNA that do not represent a problem for most applications,” says Egelhofer. “However, when scientists undertake PCR with genomic DNA from bacteria, for example, and with nonspecific primers, bacterial DNA of waterborne pathogens in the buffers or the column can impact the result. One solution is our recently launched kits, such as the QIAamp UCP Pathogen Mini kit, which possesses no detectable contamination of microbial DNA. All the materials of these kits, such as the columns and the buffers, undergo DNA decontamination processes during production, which allows us to achieve this high purity, free of nucleic acid traces.”
Amid the bewildering range of kits available, you can find what you need faster if you define the scale at which you will be purifying gDNA. “Knowing how much DNA is needed for each experiment allows the researcher to narrow down [his or her] choices to the appropriate kits,” says Kim. “The same goes for throughput. Is the researcher preparing one sample versus hundreds? The answer to this point will allow the researcher to pinpoint which kit would be the best format and to decide whether the scale is amenable to an automated workflow.”
More options are available now for increasing throughput in gDNA purification. For example, Genetic ID offers its Fast ID DNA Extraction kits in two forms: single-column formats, and more recently, 96-well plate formats. In addition to spin-column and affinity-bead formats, Zymo Research also offers several types of kits in a 96-well plate format: the ZR-96 Quick-gDNA™, the ZR-96 Quick-gDNA™ Blood, the ZR-96 Genomic DNA™-Tissue MiniPrep and the ZR-96 Viral DNA Kit™.
“It is becoming more and more common in many labs to think about automating the repetitive steps associated with isolating genomic DNA,” says Egelhofer. “There are automated systems available for many throughput needs. Systems like the QIAcube from QIAGEN automatically process the spin columns, which means that there is no change of purification chemistry required, ensuring a fast start-up and immediate results. All steps in the purification procedure are fully automated and up to 12 samples can be processed per run. Recently we also added QIAcube-specific kits that simplify the sample preparation process further, with even less hands-on time for the scientist.” For researchers who need automation in their gDNA purification, but who might also require automation with other related tasks or with other types of nucleic acids or proteins, QIAGEN offers a multipurpose system called the QIAsymphony RGQ. “This instrument automates not only the purification of the genomic DNA, RNA or proteins but also the complete workflow, from the preparation of the PCR setup through to the detection,” says Egelhofer.
Sometimes an instrument that gets you a result in the simplest, fastest way is the best tool. “Many of our customers are using our genomic DNA preparation kits for some sort of genetic analysis,” notes Kim. “In the past, genotyping was done traditionally through single nucleotide polymorphism (SNP) analysis, but this trend is leading more and more towards next-generation sequencing through platforms like our 5500 SOLiD™ systems and Ion Torrent Personal Genome Machine (PGM™) Sequencers, especially as the price per run gets cheaper. Researchers are looking to do genetic analysis with smaller sample amounts, i.e., down to the single-cell level. We are also seeing that more and more there is an emphasis on the analysis of experiments rather than the generation of data. Therefore, it has become our focus [to] help the researcher get to the end results faster with a simpler workflow.”
The image at the top of this article is from Zymo Research's ZR-Duet™ DNA/RNA MiniPrep.