ABRF 2019: Automated Genomic DNA Extraction from Large Volume Whole Blood
Content Type: Poster
Authors: Han Wei, Ph.D.; Brittany Niccum Ph.D.; Li Liu, Ph.D.; Mary Blair; Randy Pares, M.S.
Whole blood samples are critical for disease prediction and diagnosis. Obtaining intact, high quality, and concentrated genomic DNA (gDNA) from whole blood is the critical step for downstream research applications such as qPCR, microarray analysis, and next generation sequencing (NGS). The term biobank refers to a large collection of tissue samples such as blood and serum that are collected for research purposes. In order to have access to large amounts of human samples and data, an increasing number of researchers are looking to access available samples through biobanks.
Here we present an automated, high-quality, research-ready DNA extraction method from large volume blood. GenFind V3 DNA Isolation kit automated on the KingFisher Duo Prime system provides scalable and consistent system for gDNA extraction from 2 mL of whole blood. GenFind V3 DNA Isolation Kit uses SPRI (Solid Phase Reverse Immobilization) paramagnetic bead technology to isolate genomic DNA from fresh or frozen whole blood and serum containing Citrate, EDTA or Heparin anticoagulants. The combined automation and chemistry platform processing 6 samples within 2 hours (if KingFisher Presto system is preferred, 24 samples can be processed within 2 hours). A comparison between manual extraction and automated extraction was done with multiple donors. Quality metrics for gDNA yield, purity and integrity were accessed by NanoDrop and gDNA TapeStation. The gDNA yield from 2 mL whole blood was between 30-80 μg. Recovered gDNA exhibited high purity with A260/A280 ratios is about 1.8 and A260/A230 ratios is greater than 1.6. DNA integrity as measured by DNA integrity number (DIN) was above 9.0. The extract gDNA using GenFind V3 and KingFisher Duo Prime system was suitable for downstream applications such as PCR, Microarray and NGS.
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Figure 1. AMPure XP chemistry is based on SPRI paramagnetic bead technology, where the nucleic acid is immobilized on beads, contaminants washed away, then eluted. GenFind V3 DNA Isolation Kit uses SPRI paramagnetic bead technology to isolate genomic DNA from fresh or frozen whole blood and serum.
Genomic DNA Manual Extraction from 2 mL Blood using GenFind V3
Figure 2. Genomic DNA was extracted from 2 mL frozen whole blood. Total 4 different donors and three different blood collection tubes were used in the manual extraction demonstration. (A) DNA yield was measured by NanoDrop (Thermo Fisher Scientific). (B and C) DNA purity was accessed by NanoDrop (Thermo Fisher Scientific). 260/280 is about 1.8 and 260/230 is greater than 1.6 for all tested donors with three blood collection tubes: Citrate, EDTA or Heparin.
Automated Genomic DNA Extraction from 2 mL Blood using GenFind V3
Figure 3 KingFisher Duo Prime system. (A) The platform was used in GenFind V3 demonstration. (B) The magnetic head used for GenFind V3.
|DNA Purification Step||Plate Row||Reagent||Volume (μl)||Mixing Time/Mixing Speed/Pause Time||Collect Count/Time [s]||Loop|
|Lysis||Plate1-A||Blood||2000||3 min/Fast/5 min||NA||1||Heating 42°C|
|Lysis buffer LBB||1750||Slow/25 min|
|Bind||Plate 1-A||Lysate||3750||2 min/Fast/8 min||5/30 s||2|
|Bind buffer BBB||1250|
|Wash WBB-1||Plate 1-B||Wash buffer WBB||4000||1.5 min/Half/2 min/Bottom/1.5 min/Fast/3 min||5/30 s||1|
|Wash WBB-2||Plate 1-C||Wash buffer WBB||4000||1.5 min/Half/2 min/Bottom/1.5 min/Fast/3 min||5/30 s||1|
|Wash WBC-1||Plate 1-D||Wash buffer WBC||5000||1.5 min/Half/2 min/Bottom/1.5 min/Fast/3 min||5/30 s||1|
|Wash WBC-2||Plate 2-A||Wash buffer WBC||5000||1.5 min/Half/2 min/Bottom/1.5 min/Fast/3 min||5/30 s||1|
|Dry||Plate 2-A||NA||NA||1 min|
|Elution||Plate 2-B||Nuclease Free Water||400||30 s/Slow/1 min||5/30 s||1|
Table 1. KingFisher Duo Prime DNA isolation set-up
Figure 4. Genomic DNA was extracted from 2 mL frozen whole blood using KingFisher Duo System. Same cohort of samples were used in the automation extraction demonstration. (A) DNA yield was measured by NanoDrop (Thermo Fisher Scientific). The yield was higher when using automated extraction method than manual extraction. (B and C) DNA purity was accessed by NanoDrop (Thermo Fisher Scientific). 260/280 is about 1.8 and 260/230 is greater than 1.6 for all tested donors with three blood collection tubes: Citrate, EDTA or Heparin.
Beckman Coulter makes no warranties of any kind whatsoever express or implied, with respect to this protocol, including but not limited to warranties of fitness for a particular purpose or merchantability or that the protocol is non-infringing. All warranties are expressly disclaimed. Your use of the method is solely at your own risk, without recourse to Beckman Coulter. Not intended or validated for use in the diagnosis of disease or other conditions. This protocol is for demonstration only and is not validated by Beckman Coulter. © 2019 Beckman Coulter, the stylized logo, and the Beckman Coulter product and service marks mentioned herein are the trademarks or registered trademarks of Beckman Coulter, Inc. in the United States and other countries. All other trademarks are the property of their respective owners.