Kiranmai Durvasula1, Jeff Roeder1, Travis Butts1
1Omega Bio-tek, Inc.
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. In this context, Omega Bio-tek has developed a highly versatile DS line of plant DNA extraction kits encompassing both manual and automated extraction realms. The kits utilize the well-established properties of the cationic detergent, cetyltrimethylammonium bromide (CTAB), in conjunction with our proprietary silica column chemistries (manual) and magnetic bead (automated) chemistries to extract high quality DNA from a variety of plants.
Materials and Methods
To demonstrate Omega Bio-tek’s plant extraction capabilities, a variety of plant samples were subjected to automated and manual extraction performance testing. The manual extractions were performed with Omega Bio-tek’s E.Z.N.A.® Plant DNA DS Kit (D2411) spin column-based kit alongside a similar product from a leading competitor (Company Q). The automated extractions were performed using Omega Bio-tek’s automated MAG-BIND® Plant DNA DS 96 Kit (M1130) ported onto an open-ended, programmable liquid handler (Qiagen BioSprint® 96). Plant specimens used for the experiments were either acquired commercially or grown at Omega Bio-tek. All the extractions for each method tested was performed in triplicate following manufacturer’s recommended protocols. Each sample consisted of approximately 40-50 mg (wet weight) of plant leaf material and was disrupted at 1,500 rpm. The workflows for both manual and automated extractions are shown in Figure 1. Resulting purified DNA was quantified via Promega’s QuantiFluor® dsDNA system and normalized per mg of input plant material.
The DNA yields obtained following manual protocols using Omega Bio-tek’s kit was significantly higher than the leading competitor’s kit on all five different plant samples tested (Figure 2). Twenty-three of the top agricultural and biofuel crops were subjected to automated extraction using Omega Bio-tek’s M1130 kit on Qiagen’s BioSprint® 96 and compared to the manual protocol of Company Q’s.
The results shown in Table 1 reveal that the automated methods resulted in significantly higher recoveries of DNA than the competitor’s manual method on 21 of the 23 plants tested. Because the extraction methods being tested are based on different chemistries, real-time PCR for the beta-tubulin gene was performed on a subset of 100-fold dilutions of plant extracts from both the 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, or when adjusted to contain equivalent (1 ng) amounts of template (data not shown). These results indicate that matrix effects have not substantially influenced reported DNA recovery results.
Figure 2. 40-50 mg of respective fresh leaf tissue was extracted in triplicate according to the manufacturer’s recommended protocol and eluted in 100 µL. DNA analyzed with fluorescent DNA-based quantification method. Total yield was divided by total tissue amount to show ng of DNA per mg of leaf tissue.
Table 1. DNA yield comparison from 23 different plant types. 50 mg leaf sample extracted per sample according to manufacturer’s recommended protocols. DNA concentration determined via fluorescence-based nucleic acid quantification. Total yield was divided by total tissue amount to show ng of DNA per mg of leaf tissue.
Omega Bio-tek’s spin column and magnetic bead-based approaches for plant DNA extraction performed significantly better than its competitor. Overall, automated methods were much faster and required much less hands-on time than either of the manual methods. Up to 96 samples of 50 mg wet tissue (or 15 mg dry tissue) can be processed in parallel in less than 1 hour using Omega Bio-tek’s automated approach. Omega Bio-tek has leveraged their plant extraction experience and expertise to develop a robust 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™, Hamilton Firefly NIMBUS® 96, and the Thermo KingFisher™ Flex.
|E.Z.N.A.® Plant DNA DS Kit||D2411-00||5|
|E-Z 96® Plant DNA DS Kit||D1411-00||1 x 96|
|D1411-01||4 x 96|
|MAG-BIND® Plant DNA DS Kit||M1130-00||1 x 96|
|M1130-01||4 x 96|