Faith De Vergara, Application Scientist, OPS Diagnostics, LLC
Rapid advancements in sequencing methodologies have provided researchers with important analytical tools for more in-depth view of genomic studies through long read capabilities. Long read sequencing requires high molecular weight (HMW) DNA as a starting material. To avoid hurdles caused by excess mechanical and/or chemical stresses to the DNA during nucleic acid isolation processes, liquid nitrogen has traditionally been used during sample preparation in preserving the integrity of HMW DNA. However, the methods are low throughput, thus in this study different Synergy™ Plant DNA Extraction protocols were modified to isolate HMW DNA from corn leaves, using liquid nitrogen, in a high throughput, quick and safe method.
In this study we look at modifying the mechanical lysis, to reduce shearing of DNA. The modification uses liquid nitrogen, to freeze the plant samples, as the first steps in sample preparation for the Synergy™ 2.0 Plant DNA Extraction, 96 Well Synergy™ Plant DNA Extraction, and Synergy™ 1.0 Plant DNA Extraction protocols. A similar dry cryogenic homogenization method was performed by Schalamun et al. (2018) to isolate and analyze DNA fragmentation from challenging and tough leaf tissues such as Eucalyptus pauciflora with a series of cleanup steps. Based on the researcher's goal, an option to perform both short read and long read sequencing from one kit will simplify the crucial step of nucleic acid extraction and subsequent analyses.
Young leaves from Zea mays were harvested at 6 weeks.
Zea mays leaves (50 mg) and Plant Homogenization Buffer (500 µl) were added to the Synergy™ Homogenization tubes and homogenized in an HT 6™ at 4,350 rpm for 2 minutes. Tubes were centrifuged at 15,000 x g for 5 minutes, and the supernatant was transferred to a microcentrifuge tube. RNase A Solution (5 ?l) was added and incubated at 37°C for 15 minutes, then continued with the addition of 0.7 volumes of isopropanol and incubated at -20°C for 15 minutes. The solution was transferred to a spin column and centrifuged at 8,000 x g for 1 minute to bind DNA to the silica spin column, discarding the flow through. The column was washed twice with 250 ?l of ice cold 70% ethanol, centrifuged at 8,000 x g for 1 minute, with the flow through being discarded. The spin column was transferred to a clean microcentrifuge tube after the second wash step to elute DNA by adding 50 ?l of Molecular Biology Grade water and centrifuged at 15,000 x g for 1 minute.
Samples of 50 mg of fresh leaves were placed into Synergy™ Homogenization tubes and loosely capped. The tubes were submerged in liquid nitrogen for 2 minutes then homogenized using the 1600 MiniG® at 1,500 rpm for 3 minutes. Tubes were quickly centrifuged, 500 ?L of Synergy™ Plant Homogenization Buffer was added, and vortexed for 30 seconds to mix. Processed tubes then followed the rest of the Synergy™ 2.0 Plant DNA Extraction Protocol, beginning at spinning down tubes at 15,000 x g for 5 minutes to remove contaminants.
Zea mays leaves (50 mg) were homogenized in the same manner as the Cryogenic Synergy™ 2.0 Plant DNA Extraction Protocol, until the centrifugation step. Afterwards, RNase A Solution (5 ?l) was added and incubated at 37°C for 15 minutes, then continued with the addition of 0.7 volumes of isopropanol incubated at -20°C for 15 minutes. Then, the DNA was preciptated by centrifugation for 5 minutes at 15,0000 x g. The pelleted DNA was washed twice with ice cold 70% Ethanol, then pellet was dissolved by adding 50 ?l of Molecular Biology Grade Water.
Zea mays leaves (50 mg) and 350 ?l of Synergy™ Plant Homogenization Buffer were added to each well of the Homogenization Plate and tightly capped. The plate was homogenized using the 1600 MiniG® for 13 minutes at 1,500 rpm. The lysate was then centrifuged for 10 minutes at 2,100 x g, then 180 ?l of supernatant transferred to the Filter Plate with a Collection Plate under and centrifuged for 10 minutes at 2,100 x g. Each well then received 5 ?l of RNase A Solution and incubated at 37°C for 15 minutes. Then, 120 ?l of isopropanol was added to each well and incubated at - 20°C for 15 minutes. DNA was bound to the Binding Plate, with a new Collection Plate under, by transferring the solutions to the Binding Plate and spinning down for 10 minutes at 2,100 x g. DNA was washed twice with 200 ?l of ice cold 70% ethanol and spun down for 5 minutes at 2,100 x g, discarding the flow through both times. After the second wash step, the Collection Plate was replaced with an Elution Plate. DNA was eluted by adding 50 ?l of Molecular Biology Grade Water and centrifuged for 10 minutes at 2,100 x g.
Zea mays leaves (50 mg) were added to the Homogenization plate and tightly capped with the strip caps and the plate lid. The plate was submerged in liquid nitrogen for 2 minutes then homogenized in 1600 MiniG® at 1,500 rpm for 3 minutes. Plant Homogenization Buffer (350 ?l) was added to each well, and vortexed for 30 seconds. Samples were processed in accordance with the 96 Well Synergy™ Plant DNA Extraction from the initial centrifugation step of 10 minutes at 2,100 x g.
All DNA isolations were analyzed for purity using the DeNovix spectrophotometer, for quantity using the Qubit fluorometer, and size using the Agilent Bioanalyzer and by agarose gel electrophoresis.
A comparison of Synergy™ protocols with and without cryogenic homogenization showed HMW DNA can be isolated using cryogenic techniques coupled with Synergy™ CTAB isolation methods. All five methods yielded significant amounts of DNA, with good purities, however the traditional bead beating method had higher yields (Table 1). The major difference between the samples was observable when run on an agarose gel, with cryogenic samples appearing larger than the 23 Kb fragment of Lambda/HindIII digest (Fig. 2). The electropherogram images generated by the Bioanalyzer (Fig. 1, 3, and 4) indicate that the DNA is above the 17 Kb limit of the instrument.
Table 1: DNA Yield and purity as measured by Qubit (fluorescence) and DeNovix DS-11 (UV/VIS). Concentrations were measured by Qubit and Purity ratios were measured by the DeNovix. Total yields were calculated by multiplying concentration by the eluted volume.
Figure 1: (A) Gel image of DNA extraction from 50 mg of young corn leaves using the Synergy™ 2.0 Plant DNA Extraction Protocol (L1), Lambda/HindIII digest (L2), and L3-L5 are samples of cryogenic Synergy™ 2.0 Plant DNA extractions. (B) Agilent BioAnalyzer gel of cryogenic samples (bands crowd the upper purple marker at 17,000 bp).
Figure 2: (A) Agarose gel image shows a Lambda/HindIII digest in L1, six DNA preps with the Synergy™ 96-Well DNA Extraction protocol with smears in the area of 4 Kb in L3-L7. (B) Lambda/HindIII digest as a ladder in L1, negative control in L2, and six DNA preps with cryogenic Synergy™ 96-Well DNA Extraction protocol in L3-L8.
Figure 3: Depiction of gel bands of the cryogenic and standard 96 Well SYNERGY™ Plant DNA Extraction protocols on the Bioanalyzer. The tight bands between the upper purple marker of 17,000 bp and 10,380 bp for the cryogenically ground samples with the smear of the samples homogenized in buffer are between 2,000 bp and 7,000 bp.
Figure 4: (A) Synergy™ 1.0 Plant DNA Extraction protocol with the modified cryogenic protocol analyzed by agarose gel where (from left to right) DNA extractions from 50 mg of young corn are in L1-L3 and Lambda/HindIII digest in L4. (B) Same samples in Figure 4(A), ran on the Agilent BioAnalyzer with bands appearing near the upper marker of 17,000 bp.
A comparison of Synergy™ DNA extraction protocols with room temperature and cryogenic homogenization resulted in yields and fragment size are mutually exclusive. All three protocols yield at least 2 µg of genomic DNA from plant with acceptable purity ratios, i.e., 260/280 and 260/230 near 1.8 and 2.0, respectively. The Synergy™ extractions at room temperature generated more DNA, however at the expense of fragment size. For qPCR or Illumina sequencing, the basic protocol of homogenizing at room temperature works well. However, with the need for HMW DNA for Nanopore or PacBio sequencing, cryogenic homogenization followed by Synergy™ extraction produces larger fragments, but with less yield. That said, both methods produced enough DNA for sequencing. Testing with long read sequencing is ongoing, and this will be pursued further.
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