TITLE: Crop DNA Extraction with Lab-Made Magnetic Nanoparticles
AUTHORS: Haichuan Wang, Xueqi Zhao, Li Tan, Junwei Zhu, david.hyten

[DESCRIPTION]
Molecular breeding methods, such as marker-assisted selection and genomic selection, require high-throughput and cost-effective methods for isolating genomic DNA from plants, specifically from crop tissue or seed with high polysaccharides, lipids, and proteins. A quick and inexpensive high-throughput method for isolating genomic DNA from seed and leaf tissue from multiple crops was tested with a DNA isolation method that combines CTAB extraction buffer and lab-made SA-coated magnetic nanoparticles. This method is capable of isolating quality genomic DNA from leaf tissue and seeds in less than 2 hours with fewer steps than a standard CTAB extraction method. The yield of the genomic DNA was 582-729 ng per 5 leaf discs or 216-1869 ng per seed in soybean, 2.92-62.6 ng per 5 leaf discs or 78.9-219 ng per seed in wheat, and 30.9-35.4
ng per 5 leaf discs in maize. The isolated DNA was tested with multiple molecular breeding methods and was found to be of sufficient quality and quantity for PCR and targeted genotyping by sequencing methods such as molecular inversion probes (MIPs). The combination of SA-coated magnetic nanoparticles and CTAB extraction buffer is a fast, simple, and environmentally friendly, high-throughput method for both leaf tissues and seed(s) DNA preparation at low cost per sample.
The DNA obtained from this method can be deployed in applied breeding program for marker-assisted selection or genomic selection. 


[STEPS]
SECTION: Nanopartcle synthesis, sample collection, DNA preparation, evaluation and PCR
1. Weigh  FeCl2·4H2O (4.34g, 0.0218 mol).
SECTION: Nanopartcle synthesis, sample collection, DNA preparation, evaluation and PCR
2. Weigh FeCl3·6H2O (10.84 g, 0.04 mol).
SECTION: Nanopartcle synthesis, sample collection, DNA preparation, evaluation and PCR
3. Mix both FeCl2·4H2O and FeCl3·6H2O in 94 ml deionized water well in a 500 ml glass flask. (the molar ratio of Fe(II) to Fe(III) is 0.55: 1).
SECTION: Nanopartcle synthesis, sample collection, DNA preparation, evaluation and PCR
4. Adequately filter the solution with filter paper (Whatman Cat. 1440-005) to remove impurities.
SECTION: Nanopartcle synthesis, sample collection, DNA preparation, evaluation and PCR
5. Add 50 ml NaOH (5.0 M) into the solution to obtain black magnetic nanoparticles (pH = 14) in a 500 ml three-necked, round-bottomed flask provided with a magnetic stirrer.
SECTION: Nanopartcle synthesis, sample collection, DNA preparation, evaluation and PCR
6. Heat at 60 °C for 30 minutes with vigorous stirring.
SECTION: Nanopartcle synthesis, sample collection, DNA preparation, evaluation and PCR
7. Immediately slow stirring for 1 h at 80 °C under a nitrogen atmosphere for a ferrofluid containing Fe3O4 nanoparticles.
SECTION: Nanopartcle synthesis, sample collection, DNA preparation, evaluation and PCR
8. Add 5g (0.08 mole) of SA powder into the suspension (~150 mL) of magnetic nanoparticles at a molar ratio of 0.55 Fe(II): 1 Fe(III): 2 SA.
SECTION: Nanopartcle synthesis, sample collection, DNA preparation, evaluation and PCR
9. Stir consistently at 90°C for 4 h.
SECTION: Nanopartcle synthesis, sample collection, DNA preparation, evaluation and PCR
10. Separate SA coated Fe3O4 from the aqueous solution by letting round-bottomed flask sit on regular magnet for 30 minutes at room temperature.
SECTION: Nanopartcle synthesis, sample collection, DNA preparation, evaluation and PCR
11. Remove aqueous solution by pouring.
SECTION: Nanopartcle synthesis, sample collection, DNA preparation, evaluation and PCR
12. Add 250 ml pure water into flask.
SECTION: Nanopartcle synthesis, sample collection, DNA preparation, evaluation and PCR
13. Stir on stirring station for 5 minutes.
SECTION: Nanopartcle synthesis, sample collection, DNA preparation, evaluation and PCR
14. Let flask sit on regular magnet for 30 minutes at room temperature.
SECTION: Nanopartcle synthesis, sample collection, DNA preparation, evaluation and PCR
15. Repeat step 11-14 five times.
SECTION: Nanopartcle synthesis, sample collection, DNA preparation, evaluation and PCR
16. Add 250 ml pure water.
SECTION: Nanopartcle synthesis, sample collection, DNA preparation, evaluation and PCR
17. Measure pH with pH meter (Thermo Scientific Orion Star™ A215 Benchtop pH/Conductivity Meter), a neutral pH (pH 7) should be reached.
SECTION: Nanopartcle synthesis, sample collection, DNA preparation, evaluation and PCR
18. If pH is not at 7, then repeat step 11-14 2-3 more times, until pH is at 7.
SECTION: Nanopartcle synthesis, sample collection, DNA preparation, evaluation and PCR
19. Keep the final solution with SA coated magnetic particles at room temperature in a sealed glass bottle (100 ml) for future application in DNA preparation.
SECTION: Nanopartcle synthesis, sample collection, DNA preparation, evaluation and PCR
20. Collect leaf discs in ¼ inch from soybean at V2 stage, wheat at Feekes 2 stage and maize at V3 stage by using a paper puncher (Amazon, A7074005S).
SECTION: Nanopartcle synthesis, sample collection, DNA preparation, evaluation and PCR
21. 5, 10 and 15 leaf discs per collection per crop per tube (1.1 ml bullet tubes), three reps per crop.
SECTION: Nanopartcle synthesis, sample collection, DNA preparation, evaluation and PCR
22. Load the tubes on 2 ml Avan 96-well deep plate.
SECTION: Nanopartcle synthesis, sample collection, DNA preparation, evaluation and PCR
23. Add five 2 mm ceramic beads (Biospec. Cat. 11079124zx) into each tube.
SECTION: Nanopartcle synthesis, sample collection, DNA preparation, evaluation and PCR
24. Cover plate with cheese cloth and wrap with rubber band.
SECTION: Nanopartcle synthesis, sample collection, DNA preparation, evaluation and PCR
25. Turn on Virtis Freezemobile Lyophilizer (25x), temperature set at -80 °C and vacuum at 50 mT.
SECTION: Nanopartcle synthesis, sample collection, DNA preparation, evaluation and PCR
26. Load the plate with tubes on Lyophilizer (Virtis Freezemobile Lyophilizer (25x)) once the temperature and vacuum reach its setting.
SECTION: Nanopartcle synthesis, sample collection, DNA preparation, evaluation and PCR
27. Lyophilize leaf discs on Lyophilizer overnight at -80°C and 50 mT,
SECTION: Nanopartcle synthesis, sample collection, DNA preparation, evaluation and PCR
28. Remove the plate and seal tube with strip of caps for 1.2 ml micro titer tubes and store at room temperature for DNA extraction.
SECTION: Nanopartcle synthesis, sample collection, DNA preparation, evaluation and PCR
29. Collect 1, 2 and 3 dry wheat seed(s) and 1 dry soybean per 1.1 ml tube.
SECTION: Nanopartcle synthesis, sample collection, DNA preparation, evaluation and PCR
30. Add eight 2 mm ceramic beads (Biospec. Cat. 11079124zx) into each tube and seal tube with cap.
SECTION: Nanopartcle synthesis, sample collection, DNA preparation, evaluation and PCR
31. Add 500 µl extraction buffer into each tube with seed(s) and leave tubes in 4 °C fridge overnight before grinded.
SECTION: Nanopartcle synthesis, sample collection, DNA preparation, evaluation and PCR
32. Load the plate with tubes having leave tissues on the TissueLyserII (Qiagen) by using TissueLyser Adapter Set.
SECTION: Nanopartcle synthesis, sample collection, DNA preparation, evaluation and PCR
33. Shake for 30/s for 5 min.
SECTION: Nanopartcle synthesis, sample collection, DNA preparation, evaluation and PCR
34. Repeat step 24 one more time.
SECTION: Nanopartcle synthesis, sample collection, DNA preparation, evaluation and PCR
35. Remove tubes from tissuelyser.
SECTION: Nanopartcle synthesis, sample collection, DNA preparation, evaluation and PCR
36. Spin down at 3700 rpm for 30 sec.
SECTION: Nanopartcle synthesis, sample collection, DNA preparation, evaluation and PCR
37. Add 500 µl of DNA extraction buffer (Tris-HCl 50 mM, pH 6.5, CTAB 1%, EDTA 10 mM, beta-mercaptoethanol 5%, 1.4 M NaCl and 2% PVP-40) into each tube.
SECTION: Nanopartcle synthesis, sample collection, DNA preparation, evaluation and PCR
38. Seal tubes with strip of caps for 1.2 ml tubes well
SECTION: Nanopartcle synthesis, sample collection, DNA preparation, evaluation and PCR
39. Incubated plate with tubes having lyophlized leaf tissues at 65°C for 60 min on a water bath.
SECTION: Nanopartcle synthesis, sample collection, DNA preparation, evaluation and PCR
40. Clean the water on plate by using paper towel.
SECTION: Nanopartcle synthesis, sample collection, DNA preparation, evaluation and PCR
41. Centrifuge at 3700 rpm for 15 min at 25°C.
SECTION: Nanopartcle synthesis, sample collection, DNA preparation, evaluation and PCR
42. For grinding the seeds in tubes, follow the step 32-36 and step 38-41.
SECTION: Nanopartcle synthesis, sample collection, DNA preparation, evaluation and PCR
43. Take a new a new 0.2 ml 96-well non-skirt plate, add 2 µl beads (60 ng/µl) and 1 µl of RNase A (1 µg/µl) into each well bottom.
SECTION: Nanopartcle synthesis, sample collection, DNA preparation, evaluation and PCR
44. Transfer 100 µl supernatant per tube into well, one tube into one well.
SECTION: Nanopartcle synthesis, sample collection, DNA preparation, evaluation and PCR
45. Mix well by pipetting up and down 3 times and leave plate on bench for 10 min at room temperature.
SECTION: Nanopartcle synthesis, sample collection, DNA preparation, evaluation and PCR
46. Add 100 µl isopropanol into each well.
SECTION: Nanopartcle synthesis, sample collection, DNA preparation, evaluation and PCR
47. Mix well by pipetting up and down three times.
SECTION: Nanopartcle synthesis, sample collection, DNA preparation, evaluation and PCR
48. Leave plate on bench for 10 min at room temperature.
SECTION: Nanopartcle synthesis, sample collection, DNA preparation, evaluation and PCR
49. Let the plate sit on a magnet (dynaMag-96 side) for 5 min.
SECTION: Nanopartcle synthesis, sample collection, DNA preparation, evaluation and PCR
50. Remove all solution carefully from each well by pipetting. don't touch beads pellet on wall.
SECTION: Nanopartcle synthesis, sample collection, DNA preparation, evaluation and PCR
51. Add 100 µl 80% EtOH without disturbing the beads.
SECTION: Nanopartcle synthesis, sample collection, DNA preparation, evaluation and PCR
52. Wait for 30 sec.
SECTION: Nanopartcle synthesis, sample collection, DNA preparation, evaluation and PCR
53. Remove EtOH by pipette and air dry for 5 min.
SECTION: Nanopartcle synthesis, sample collection, DNA preparation, evaluation and PCR
54. Remove 96-well PCR plate from magnet.
SECTION: Nanopartcle synthesis, sample collection, DNA preparation, evaluation and PCR
55. Add 50 µl 10 mM Tris-HCl (pH 8).
SECTION: Nanopartcle synthesis, sample collection, DNA preparation, evaluation and PCR
56. Mix well with a pipette or slightly vortex.
SECTION: Nanopartcle synthesis, sample collection, DNA preparation, evaluation and PCR
57. Spin down the plate.
SECTION: Nanopartcle synthesis, sample collection, DNA preparation, evaluation and PCR
58. Leave plate on bench for 10 min at room temperature (not on magnet).
SECTION: Nanopartcle synthesis, sample collection, DNA preparation, evaluation and PCR
59. Place plate back on the magnet for 3-5 min.
SECTION: Nanopartcle synthesis, sample collection, DNA preparation, evaluation and PCR
60. Transfer solution (~ 50 µl) with DNA into a new PCR plate.
SECTION: Nanopartcle synthesis, sample collection, DNA preparation, evaluation and PCR
61. Leave the plate on ice.
SECTION: Nanopartcle synthesis, sample collection, DNA preparation, evaluation and PCR
62. Measure DNA concentration on Qubit (2.0) by following kit instructions (Qubit™ dsDNA Quantification Assay Kits, Q32850).
SECTION: Nanopartcle synthesis, sample collection, DNA preparation, evaluation and PCR
63. Load 1 µl of DNA/sample/well on 1.5% agarose gel with SYBR. Lambda DNA used as DNA marker.
SECTION: Nanopartcle synthesis, sample collection, DNA preparation, evaluation and PCR
64. Label the plate, seal plate with foil plate seal (Thermo Scientific, Cat.232698) and store plate at -20°C freezer for future use.
SECTION: Nanopartcle synthesis, sample collection, DNA preparation, evaluation and PCR
65. Run at 120 V for 45 min in 1x TBE buffer.
SECTION: Nanopartcle synthesis, sample collection, DNA preparation, evaluation and PCR
66. Evaluate the gel under UV Transilluminator (UVP) at 488 nm wavelength.
SECTION: Nanopartcle synthesis, sample collection, DNA preparation, evaluation and PCR
67. Create a daughter plate to a final concentration of 12 ng/µl/DNA sample for further evaluation by molecular inversion probe (MIP) and conventional PCR analysis, respectively.
SECTION: Nanopartcle synthesis, sample collection, DNA preparation, evaluation and PCR
68. Store the plate with all DNA samples in -20 °C freezer for use.
SECTION: Nanopartcle synthesis, sample collection, DNA preparation, evaluation and PCR
69. Mix 5 µl of 2x master mix (BioRad, Cat. 172-5310), 0.25 µl of 10 µM primers (Forward and Revers primer respectively) and 1 µl genomic DNA in a 200 µl PCR tubes (Axygen, Cat. PCR02C) at a final reaction volume of 10 µl/sample.
SECTION: Nanopartcle synthesis, sample collection, DNA preparation, evaluation and PCR
70. Conduct PCR with following conditions, 98 °C/30 s, 21x (98 °C/10 s, 60 °C/30 s, 72 °C/30 s), 72 °C/60 s, holding at 12 °C on BioRad C1000 thermal cycler.
SECTION: Nanopartcle synthesis, sample collection, DNA preparation, evaluation and PCR
71. Load 3 µl of DNA/sample/well on 1.5% agarose gel with SYBR along with 100 bp DNA ladder.
SECTION: Nanopartcle synthesis, sample collection, DNA preparation, evaluation and PCR
72. Run at 120 V for 45 min in 1x TBE buffer.
SECTION: Nanopartcle synthesis, sample collection, DNA preparation, evaluation and PCR
73. Evaluate the gel under UV Transillumnator at 488 nm wavelength.
SECTION: Molecular Inversion Protocol (MIP)
75. Dilute 998 individual probe synthesized by Integrated DNA Technologies (Coralville, IA, USA) at 0.2 nmole (Supplemental Table S2, Wang et. al. 2022) with nuclease free water at 1:10.
SECTION: Molecular Inversion Protocol (MIP)
76. Pool 2 μl each probe together in a 2 ml centrifuge tube.Phosphorylate the pooled probes in 200 μl PCR tube in a 20 μl reaction consists of 15 μl of diluted Pooled probes, 1× T4 DNA ligase buffer, 1 mM ATP, and 0.5 U of T4 polynucleotide kinase (ThermoFisher, T4 polynucleotide kinase kit).
SECTION: Molecular Inversion Protocol (MIP)
77. Close the cap and mix well.
SECTION: Molecular Inversion Protocol (MIP)
78. Spin down.
SECTION: Molecular Inversion Protocol (MIP)
79. Load the PCR tube to thermal cycler and incubate at 37 °C for 30 min followed by an inactivation of the kinase at 75 °C for 10 min.
SECTION: Molecular Inversion Protocol (MIP)
80. Spin down tube and leave tube on ice in an ice bucket. Make a master mix for hybridization and extension, which consists of 1.05 μl pooled phosphorylated
SECTION: Molecular Inversion Protocol (MIP)
81. probes (∼1,000:1 DNA to probe ratio), 1.25 μl 10× Ampligase DNA ligase buffer (Epicentre Technologies), 1 μl of 5 M Betain (Sigma Aldrich), 3.25 U AmpliTaq DNA polymerase (ThermoFisher), 16 U of DNA Ampligase (Epicentre Technologies), 0.016 mM dNTP, and water. Note, this is for one reaction/well. Scale it up accordingly with your sample size.
SECTION: Molecular Inversion Protocol (MIP)
82. Take a new 384-well plate.
SECTION: Molecular Inversion Protocol (MIP)
83. Add 5.25 μl at 12 ng/ul (63 ng) DNA sample into corresponding well. One sample/well.
SECTION: Molecular Inversion Protocol (MIP)
84. Dispense the master mix from step 8 into each well by using Mantis Liquid Handler (Formulatrix). The final reaction volume is 12.5 µl per reaction per sample per well.
SECTION: Molecular Inversion Protocol (MIP)
85. Seal the 384-well plate with film by using PCR Plate Sealer and mix well by vortexing.
SECTION: Molecular Inversion Protocol (MIP)
86. Spin down.
SECTION: Molecular Inversion Protocol (MIP)
87. Leave plate on ice.
SECTION: Molecular Inversion Protocol (MIP)
88. Turn on the thermal cycler.
SECTION: Molecular Inversion Protocol (MIP)
89. Load the plate to thermal cycler.
SECTION: Molecular Inversion Protocol (MIP)
90. Run a program as following parameters. 98 °C for 10 min at 60 °C for ∼24 h. Note, the heated lid for the PCR machine is set to 105 °C for the first 30 min and then changed to 80 °C after the first 30 min of the incubation.
SECTION: Molecular Inversion Protocol (MIP)
91. Stop PCR reaction and remove the plate.
SECTION: Molecular Inversion Protocol (MIP)
92. Spin down plate, then leave the plate on ice.
SECTION: Molecular Inversion Protocol (MIP)
93. Remove the sealing film.
SECTION: Molecular Inversion Protocol (MIP)
94. Dispense 2 μl mix of 6.4 U of Exo I (New England Biolabs), 32 U of Exo III (New England Biolabs), 0.2 μl 1× Ampligase DNA ligase buffer into each well using a Mantis Liquid Handler (Formulatrix).
SECTION: Molecular Inversion Protocol (MIP)
95. Seal plate with seal film using PCR Plate Sealer, mix plate well by vortexing and spin down.
SECTION: Molecular Inversion Protocol (MIP)
96. Load the plate to thermal cycler and incubate at 37 °C for 30 min followed by 95 °C for 2 min to digest uncircularized MIPs and DNA templates.
SECTION: Molecular Inversion Protocol (MIP)
97. Remove the plate from thermal cycler, spin down and leave it on ice.
SECTION: Molecular Inversion Protocol (MIP)
98. Make a PCR master mix 2.5 μl of 2× iProof HF Master Mix (BioRad) and 0.75 μl water in a 200 μl PCR reaction tube. (Note, this volume is for one reaction per well, you need to scale up volume accordingly with your sample size).
SECTION: Molecular Inversion Protocol (MIP)
99. Close the cap mix well by vortexing.
SECTION: Molecular Inversion Protocol (MIP)
100. Spin down and leave tube on ice.
SECTION: Molecular Inversion Protocol (MIP)
101. Take a new 384-well PCR plate.
SECTION: Molecular Inversion Protocol (MIP)
102. Add 0.5 μl of 10 μM indexed forward and reverse primers (Supplemental Table S2) per well.
SECTION: Molecular Inversion Protocol (MIP)
103. Transfer 1.25 μl of circularized MIP capture taken from step 24 as template by using multichannel pipette.
SECTION: Molecular Inversion Protocol (MIP)
104. Dispense 3.75 μl master mix from step 27 into each well by using Mantis.
SECTION: Molecular Inversion Protocol (MIP)
105. Seal plate with seal film, vortex slightly to mix well.
SECTION: Molecular Inversion Protocol (MIP)
106. Spin down the plate.
SECTION: Molecular Inversion Protocol (MIP)
107. Load the plate to the thermal cycler.
SECTION: Molecular Inversion Protocol (MIP)
108. Run PCR by following conditions: 98 °C for 30 s; 21 cycles of 98 °C for 10 s, 60 °C for 30 s, 72 °C for 30 s;and 72 °C for 60 s, holding at 12 °C.
SECTION: Molecular Inversion Protocol (MIP)
109. Spin down the plate.
SECTION: Molecular Inversion Protocol (MIP)
110. Remove the seal film.
SECTION: Molecular Inversion Protocol (MIP)
111. Pool PCR reactions by taking 1 μl/well/sample in a 1.5 ml centrifuge tube.
SECTION: Molecular Inversion Protocol (MIP)
112. Mix well and spin down, then leave tube on ice.
SECTION: Molecular Inversion Protocol (MIP)
113. Take 30 μl pooled PCR products from step 39 and separate it on 1.5% agarose gel along with 50-bp (Fisher) and 100-bp DNA ladders (GoldBio) for 25-30 min at 120 V.
SECTION: Molecular Inversion Protocol (MIP)
114. Stain the gel with 1x SYBR in running buffer for 5-10 min.
SECTION: Molecular Inversion Protocol (MIP)
115. Excise the sharp band at ∼286 bp by using clean razor blade.
SECTION: Molecular Inversion Protocol (MIP)
116. Purify gel from step 42 with gel extraction kit (Qiagen) by following the manufacturer's instructions.
SECTION: Molecular Inversion Protocol (MIP)
117. Quantify the purified PCR products on Qubit (ThermoFisher) with Qubit DNA HS Assay Kit (ThermoFisher).
SECTION: Molecular Inversion Protocol (MIP)
118. Quantify purified pooled PCR products further precisely by using the KAPA library quantification kit Illumina platforms (KAPA, KK4824) on a QuantStudio 6 Flex system (Thermo Fisher Scientific).
SECTION: Molecular Inversion Protocol (MIP)
119. Sequence the purified PCR products on NextSeq 550/500 system (Illumina) at 150 bp single read with customer specific sequencing primers (Supplementary Table S3).
SECTION: Molecular Inversion Protocol (MIP)
120. Filter the raw reads, including remove adapter sequence contamination, truncated reads, and reads with overall low base quality the raw reads with Trimmomatic (software).
SECTION: Molecular Inversion Protocol (MIP)
121. Aligned the filtered reads to a custom reference genome, which consists of 200-bp flanking regions around each targeted SNP for each of the 998 SNPs using Bowtie 2 in end-to-end mode with the sensitive setting.
SECTION: Molecular Inversion Protocol (MIP)
122. Discard reads with a mapping quality score of 20.
SECTION: Molecular Inversion Probe (MIP)
74. Switch to MIP protocol