SYNERGY™ CTAB-based DNA isolation in a 96 well format was evaluated using a glass-fiber filter plate capture method versus silica-coated magnetic nanoparticles (MNPs). DNA was isolated from the leaves, stems, and roots of six-week-old maize plants by bead beating using a SYNERGY™ protocol, followed by purification using a 96 well glass-fiber filter plate or MNPs using an IsoPure™ automated extraction system. Isolated DNA was subsequently analyzed by gel electrophoresis, Bioanalyzer, optical density, and qPCR. Yields from the two processes differed significantly with roots and stems, with MNPs performing significantly better than the filter plate. Yields from leaf tissue between the two methods were comparable, with the filter plate producing slightly higher concentrations. Purity of DNA isolated from roots and stems, as measured by 260/280 ratio, were better for MNPs than filter plates, while leaf tissue was the same.
In isolating nucleic acids from plants, SYNERGY™ isolation chemistry using CTAB has been found to be a simple and cost-effective alternative to guanidine-based kits (Jeffrey et al., 2021). Traditional CTAB buffers have been widely used for plant DNA isolation as the homogenization buffers precipitate polysaccharides and inhibit polyphenol oxidase. With SYNERGY™, CTAB buffers are used in conjunction with a grinding resin that also serves as the first step of the purification process. Lysates generated from bead beating still rely on either nucleic acid precipitation or binding and elution from silica membranes to complete the purification.
With the increasing number of samples being processed in diagnostic and research laboratories, silica membranes are being replaced by silica-coated magnetic nanoparticles (Tang et al., 2020). Correspondingly, the SYNERGY™ isolation process has been modified for use with MNPs. The SYNERGY™ 3.0, is available, includes MNPs as subsequent purification of CTAB based SYNERGY™ lysates. The kit is designed for manual extractions and substitutes MNPs for spin columns. In theory, manual MNP protocols should easily adapt to automated systems. The SYNERGY™ chemistry was adapted to high throughput processing, using an IsoPure™ automated processing instrument and samples homogenized in a 96 well SYNERGY™ plate. The high throughput process was compared to the manual filter plate protocol.
The goal of this study is to compare filter plate isolation to magnetic nanoparticle-based DNA isolation. Maize root, stem, and leaf samples are homogenized in a SYNERGY™ 96-well plate, and the lysates processed manually and with an IsoPure™ Mini automated system. Isolated DNA was analyzed by gel electrophoresis, Bioanalyzer, UV spectroscopy, and qPCR of the maize actin gene.
Materials and Methods
Sample Processing: Six-week-old maize plants were extracted from planters with roots intact. The plants were washed with deionized water to remove residual soil. Four roots, six stems, and six leaf samples were cut and weighed for processing (Table 1). Each sample was added to a well of a 96 well SYNERGY™ homogenization plate along with 350 µl of CTAB homogenization buffer and then capped. Samples were processed in a 1600® Mini-G (SPEX SamplePrep, New Jersey, USA) for 10min at 1,500 rpm. Followed by centrifugation for 10 min at 2,100 x g. Eight lysates from two roots, three stems, and three leaves) were used in MNP-based isolation while the other eight samples were used in a filter plate-based isolation following the 96 well SYNERGY™ Plant DNA extraction kit protocol.
Table 1: Average sample mass (mg) for maize root, stem, and leaf
MNP Isolation: An IsoPure™ Mini (BM-AP1016) extraction system was used to isolate DNA. Two columns (16 samples) can be processed in a run. The processing steps are the same as a full IsoPure™ instrument that handles a full 96 well plate. Samples and reagents are loaded in columns of a 96 well plate (samples are in rows 1 and 7, with each of the remaining two sets of five rows being used for reagents). The plate was loaded as follows:
Columns 1 and 7 – 5 µl RNase A (SYNERGY™ kit),
Columns 2 and 8 - 25 µl of SYNERGY kit MNPs,
Columns 3 and 9 – 500 µl wash buffer VF1,
Columns 4 and 10 – 500 µl wash buffer VF2,
Columns 5 and 11 – empty, and
Columns 6 and 12 – 100 µl elution buffer EB1.
Cleared lysate from the homogenization plate (approximately 150 to 170 µl) was transferred to columns 1 and 7 of the extraction plate. The plate was placed in the IsoPure™ Mini and heated to 37°C for RNase A treatment time was 10 min. The plate was removed and 400 µl of ethanol was added to columns 1 and 7. The plate was returned to the IsoPure™ Mini where the MNPs were retrieved from columns 2 and 8 with magnetic pins and transferred to columns 1 and 7, respectively. This was mixed for 5 min. allowing for the DNA to bind to the MNPs. The beads were retrieved and transferred to columns 3 and 9 for the first wash. After 1 minute the MNPs were collected and transferred to columns 4 and 10 for the second wash. After another minute, the MNPs were collected and allowed to air dry for 10 minutes and then dropped into columns 6 and 12, were preheated to 70°C to elute the DNA. The beads were removed after 5 min., leaving the eluted DNA in columns 6 and 12.
The eluted DNA in columns 6 and 12 was used for analysis.
Filter Plate: After homogenization and centrifugation, 180 µl of the lysate from the strip was transferred to the first column of the filter plate. The filter plate was placed on top of a collection plate and centrifuged at 2,100 x g for 10 min to remove residual debris. The cleared lysate was treated by adding 5 µl of RNase A (10 mg/ml) and incubated at 37°C for 15 min. Isopropanol (120 µl) was added to each well, mixed and incubated at -20°C for 15 min. A glass fiber binding plate was placed on top of a collection plate and the samples were transferred to the binding plate. The plate is centrifuged for 10 min at 2,100 x g and the flowthrough was discarded. The filters were washed with 200 µl of ice cold 70% ethanol and centrifuged for 5 min at 2,100 x g. This process was repeated once. For elution, 100 µl of molecular biology grade water was added to the plate and then centrifuged for 10 mins at 2,100 x g.
Electrophoretic Analysis: Agarose gel electrophoresis was used to assess qualitative yields and fragment size from DNA samples. Pre-cast 1% SeaKem Agarose gels in 1X TAE buffer (Lonza) were loaded with 5 µl sample mixed with 2 µl loading buffer. The gels were electrophoresed for 25 minutes at 96V. DNA was visualized on a UV transilluminator and photographs taken with an iPhone. Samples were also analyzed with a Bioanalyzer (Agilent) using the DNA 12000 Reagents and a DNA chip. The analysis plot contained fragment size and quantitated the amount of DNA using fluorescence. The chip was loaded according to the directions outlined by Agilent with 1 µl of sample per well.
Absorbance: The DS-11 Spectrophotometer (DeNovix, Delaware, USA) was used to estimate concentration and purity of the DNA samples. The dsDNA app on the spectrophotometer automatically calculates 260/280 ratio and concentration from 1 µl samples.
Polymerase Chain Reaction (PCR): To assess the carryover of any potential inhibitors, qPCR was used to detect the maize actin gene. Cycle thresholds (Ct) should be relative to the concentrations of DNA isolated in each sample. The reaction for this experiment included actin primer (Sequence: [FWD] 5’ - GCC ACG TAC AAC TCC ATC AT – 3’; [REV] 5’ – GAC GTG ATC TTG CTC ATA C – 3’), using SYBR Green for detection.
Results and Discussion
The capture of maize DNA using MNPs was generally more effective than using the filter plate. Gel electrophoresis (Fig. 1) shows clear and relatively robust band/smears for MNPs, while only visible DNA in the leaf samples for the filter plates. The exception is the filter plate isolated leaf samples, which indicates significant concentrations of DNA. The alternative analysis using the Bioanalyzer (Fig. 2), which uses fluorescence and hence greater sensitivity, shows DNA in all samples, though MNP samples have much greater yield. The size of the fragments isolated, i.e., 2-5 Kb, is also consistent with DNA isolated by bead beating. The beads create significant shearing forces and reduce typically large fragments to a size range that falls around 4 kb.
Figure 1: Agarose gel electrophoresis of MNP and filter plate isolated maize DNA.
Figure 2: Bioanalyzer electropherogram of maize DNA samples.
It is noteworthy that both isolation processes had significant yield with leaves. The concentration of DNA for leaves is higher than roots and stems due to the DNA associated with chloroplasts while cell density may also be higher in actively growing leaves (Sakamoto and Takami, 2018). The higher yields may be more a function of mass action, i.e., high concentrations of DNA sticking to the filters and saturating the MNPs, than performance of the filter plates. The static nature of the filters, in that they are fixed and require lysate to be pushed through pores, should be kinetically less efficient than the homogeneous nature of MNPs in solution. The fact that the two processes are about equal may be related to overloading the purification processes.
Figure 3 shows yields as measured by the Bioanalyzer. The yields correlate to the gel electrophoresis and Bioanalyzer results.
Figure 3: Yields as determined by fluorescence measured by Bioanalyzer.
Absorbance measurements were used to estimate purity of the DNA. Ideal ratios equal 1.8, and MNP samples landed very close to this target. Filter plate roots and stems were above and below, respectively, which may be caused by inadequate washing (Fig. 4). Once again, the homogeneous nature of handling MNPs promotes good exchange of buffers and helps to eliminate carryover. Wash buffer VF1 is guanidine based, which adsorbs more strongly at 260 than 280, which can lead to higher ratios. RNA may also cause higher ratios. The lower ratio may be caused by residual protein not adequately removed by washing the filter plate.
Figure 4: Absorbance and 260/280 ratios of MNP and filter plate samples.
Quantitation of maize actin gene by qPCR displays less dramatic differences in yield than the above measurements. Indeed a few cycles difference between samples can represent 10 to 100 times differences target DNA concentration. In all samples, the Ct values correlate to the relative mass or gel estimations of yield. The important aspect of this analysis is that both methods yielded PCR amplifiable DNA.
Figure 5: Ct values for maize actin qPCR of MNP and filter plate samples.
SYNERGY™ chemistry for the isolation of DNA from maize has been shown to be effective, often with difficult to process plants. The option of using MNPs for the final steps of the purification has been shown to be highly effective. Consequently, the product can be used for high throughput laboratories requiring quality DNA and lower processing costs.
The data shows the quality of DNA and DNA yield is better with the magnetic nanoparticle isolation for the stem and root samples. For the leaf samples, the yields and quality are not significantly different. For researchers working with samples from roots or stems the magnetic nanoparticle-based DNA extraction is a better option than the filter plate. Another benefit of magnetic nanoparticle-based extraction is using automation, such as the KingFisher or IsoPure™ Mini, which minimizes the laborious nature of DNA purifications.
Overall, the automated magnetic nanoparticle-based plant DNA extraction provides a more consistent, more efficient, and less labor-intensive isolation across multiple plant sample types compared to using a 96-well filter plate. Though initial costs in instrumentation can be significant, DNA quality and labor savings make this approach worth pursuing.
Jeffrey, Luke C. et al. 2021. “Bark-Dwelling Methanotrophic Bacteria Decrease Methane Emissions from Trees.” Nature Communications 12(1): 2127.
Sakamoto, Wataru, and Tsuneaki Takami. 2018. “Chloroplast DNA Dynamics: Copy Number, Quality Control and Degradation.” Plant and Cell Physiology 59(6): 1120–27.
Tang, Congli et al. 2020. “Application of Magnetic Nanoparticles in Nucleic Acid Detection.” Journal of Nanobiotechnology 18(1): 62.