The incredible diversity of the plant kingdom comes with a wide range of DNA extraction challenges. These challenges include extensive variations in plant structures and in their chemical composition of polysaccharides, polyphenolic compounds and humic substances, all of which can interfere with extraction efficiency and/or inhibit downstream applications.
Omega Bio-tek has long appreciated the challenges unique to plants and has worked with many agricultural customers to develop extraction chemistries, protocols and multi-sized kits to optimize specific plant research workflows. Because no single extraction method suits all, Omega Bio-tek has developed a variety of lysis, washing and DNA binding buffers to work with our assortment of proprietary magnetic bead and silica column chemistries to address plant extraction challenges.
To demonstrate Omega Bio-tek's plant extraction capabilities and to assist customer selection of the proper extraction chemistry for their plants of interest, equivalent starting amounts of leaf material from 23 of the top agricultural and biofuel crops were subjected to automated and manual extraction performance testing with four different kits. The automated extractions were performed using two of Omega Bio-tek's automated Mag-Bind® plant DNA extraction kit chemistries ported onto a programmable, open robotic liquid handler. The manual extractions were performed with Omega Bio-tek's E.Z.N.A.® spin column-based plant extraction kit alongside a similar product from a leading competitor.
The results in Table 1 reveal that the automated methods resulted in significantly higher recoveries of DNA than either of the manual methods tested. Of course, the automated methods were also much faster and involved much less hands-on time than the manual methods. Interestingly, depending on plant type, maximum DNA yields were obtained by either of the two different Omega Bio-tek automated extraction technologies. For the manual extraction methods, Omega Bio-tek's more cost-effective product performed on par with the leading competitor product with varying plant type-dependent results. Altogether, these results demonstrate the benefit of Omega Bio-tek's multiple technical approaches for plant DNA extraction.
|Omega Bio-tek||Company Q|
|Table 1. Amounts of purified DNA extracted using four different extraction kits. Higher performance in DNA yields (ng/mg tissue) highlighted in green.|
Figure 1. Bar graph representing the average real-time PCR cycle threshold value from 3 separate real-time PCR reactions of a 0.01 dilution of the pepper plant leaf extracts obtained from each of four different extraction methods. The measured quantity (ng) of DNA in each extract is shown in parentheses above the associated column. A no-template control (NTC) reaction is included.
Figure 2. Bar graph representing the average real-time PCR cycle threshold value from 3 separate real-time PCR reactions. 1.0 ng of DNA from the cotton plant leaf extracts as obtained from four different extraction methods. A no-template control (NTC) reaction is included.
Because the extraction methods being tested are based on different chemistries, it is possible that differences in the chemical composition of the resulting extracts might adversely influence DNA quantification, otherwise known as "matrix effects." To ensure that the DNA quantity measurements were accurate, real-time PCR from the beta-tubulin gene was performed on a subset of 100-fold dilutions of plant extracts from all four methods. The differences in the resulting real-time PCR cycle threshold values were consistent with the amount of template measured to be present in each extract (Figure 1), or when adjusted to contain equivalent (1 ng) amounts of template (Figure 2). These results indicate that matrix effects have not substantially influenced reported DNA recovery results.
Plant specimens were acquired commercially or grown at Omega Bio-tek. Each extraction of each plant type was performed in triplicate for each method tested. Each sample consisted of approximately 50 mg (wet weight) of plant leaf material and was disrupted by bead-beating via a Geno/Grinder 2010 for 2 minutes at 1,500 RPM. DNA was extracted using Omega Bio-tek's automated technologies (product numbers M1128 [Mag-Bind® Plant DNA Plus 96 Kit] and M1130 [Mag-Bind® Plant DS 96 DNA Kit]) ported onto an open and programmable automated liquid handler (Qiagen BioSprint 96). Manual extractions were performed using Omega Bio-tek spin column technology (product number D5511, E.Z.N.A.® SP Plant DNA Kit) versus an extraction kit from a leading competitor (Company Q) following manufacturer's instructions. Resulting purified DNA was quantified via Promega's QuantiFluour DS DNA system and normalized per mg of plant material input. Real-time PCR was performed with Agilent's Brilliant III 2X SYBR mix and primers specific for the beta-tubulin gene using a standard amplification protocol on the ABI 7900.
Omega Bio-tek has leveraged their plant extraction experience and expertise to develop a robust and multi-tiered solution for plant DNA extraction needs. Options are available for automation on open liquid handling and magnetic processor platforms such as the Hamilton Microlab STAR and Thermo KingFisher Flex.
|Kit Name||Product No.||Preps|
|E.Z.N.A.® SP Plant DNA Kit||D5511-01||50|
|Mag-Bind® Plant DNA DS Kit||M1130-00||1 x 96|
|M1130-01||4 x 96|
|Mag-Bind® Plant DNA Plus Kit||M1128-00||1 x 96|
|M1128-01||4 x 96|