by Laura Lane
Methods for genomic DNA purification have come a very long way since 1869 when Johann Friedrich Miescher first isolated what he called nuclein. Eventually identified as DNA, the construct of nucleic acids has taken center stage in the life sciences and, increasingly, in other disciplines such as forensic, agricultural and environmental sciences.
“People are studying anything that they can get DNA from, including some complex environmental samples like soil and water,” says Marc Van Eden, PhD, vice president of business development/marketing at Zymo Research. “That presents a whole new bag of considerations as it pertains to effective sample processing.”
Chief among considerations is the quality of DNA that dictates the success of subsequent analysis. Unlike the days of Miescher, researchers now demand DNA that’s free of contaminants, such as salts, ionic detergents, alcohols and phenol, which could interfere with downstream applications by inhibiting enzymatic reactions. Sometimes these substances are introduced during the extraction process. Other times, such as in the case of forensics, the contaminants are introduced during collection of the original sample.
“Almost anyone can purify DNA,” he says. “But the question remains; can they purify DNA that is well-suited for enzymatic manipulation?”
For many researchers, the principal enzyme of concern is DNA polymerase and its function in PCR. Certain substances can co-elute with the DNA during the purification process that can interfere directly with DNA polymerase or with its cofactors, such as magnesium, which the enzyme depends on to function properly. Or, inhibition occurs when the contaminants bind to single- or double-stranded DNA involved with amplification. Other applications that are vulnerable to inhibitors include DNA methylation analysis, which hinges upon the action of methylases, and gene sequencing.
Fortunately, the market now offers a wide variety of options for obtaining pure DNA. No matter what type of sample you’re working with, you’re sure to find something that suits your needs.
But if you’re interested in using the latest products, don’t go looking for those based on phenol and chloroform, which has long been the workhorse for DNA extraction. Sold under brand names such as TRI Reagent or TRIzol, the reagents can linger in the resulting product and require hours of hands-on labor.
“Companies are moving toward non-hazardous extractions,” says Emmy Leung, technical and marketing specialist at Wako Chemicals USA, pointing out the hazards that phenol and chloroform pose on the health and environment.
Wako’s extraction protocols depend on a method based on sodium iodide that acts as a chaotropic agent. The approach allows researchers to avoid hazardous substances. Wako also has a new kit that aids in reducing the amount of DNA that’s oxidized – and therefore compromised – during the extraction process.
But it’s not all about the reagents. Hardware is important too. For genomic DNA purification, spin columns remain the most popular, although other technologies such as magnetic beads are slowly becoming more prevalent. While companies offer different designs, most hinge on DNAs affinity for binding to silica, which is usually built into the spin column as a silica membrane. The force of a vacuum or centrifuge helps to wash the sample and prepare it for elution.
To choose between all the different options available, simply look for those that match your project parameters. What type of samples are you starting with? How much DNA will you be extracting? What is your level of throughput? Do you want automated processing? Just when you might have something picked out, the choices widen.
For example, soon, Zymo Research will release a micro-elution column that’s specially designed to aid in the purification of large-sized DNA, or strands with more than 150,000 base pairs. Typical extraction processes depend on centrifugation with shearing forces that can tear apart genomic DNA. “We’re using non-invasive ways in order to ensure purification without shearing forces,” Van Eden says.
You’ll also find an increasing number of solutions for extracting genomic DNA from formalin-fixed, paraffin-embedded tissues, such as the QIAamp DNA FFPE Tissue Kit from Qiagen. Traditionally, this endeavor has been wrought with frustration as collection and fixation methods have degraded the DNAs quality.
PreAnalytix, a Qiagen/BD company, provides an innovative solution to this problem with their new PAXgene Tissue System. Launched in June of this year, the system includes everything you need to collect and fix samples for preserved tissue morphology while also stabilizing the integrity of genetic material. Then, by using the PAXgene Tissue spin column kits, you can extract and purify DNA, RNA or miRNA from the preserved tissues.
Whichever columns you choose, you’re likely to find them also available in a format ready for a high throughput pace. You can opt for 96-well formats, which essentially function as 96 columns fused together. Or, you can look into purchasing automated instruments, many of which feature a compact, desktop design.
“People are now looking for completely automated systems to set up the sample prep, so they can go away and focus on other parts of their research,” says Janina Schaper, PhD, global product manager at Qiagen.
For lower throughput, Qiagen’s QIAcube could be the ticket. It simultaneously handles 12 samples. QIASymphony serves the need for medium throughput, processing 24 to 96 samples. Higher throughput machines, which can process hundreds of samples at the touch of a button, also abound.
Automation or not, today’s genomic DNA purification kits offer plenty of convenience and confidence in the finished product.