APPLICATION BENEFITS
DNA extraction is an essential step in many molecular biology workflows. Purification of high-quality, nucleic acids is key for successful downstream applications such as DNA sequencing, amplification, and genotyping. Effective and efficient sequence detection requires preparation methods that isolate and purify the target of interest from crude lysates and other complex biological samples.
SOLUTIONS
ESP™ magnetic bead technology purifies samples faster than resins and columns. The revolutionary, slide-based extraction gently moves your samples through isolation with a simple slide of the handle. The ergonomic device is easy to operate and delivers rapid purification of samples with high yield and without crossover contamination.
By Mary Regier | GILSON, INC.; Middleton, WI, USA | Scott Barry and David J Gluckenberger | SALUS DISCOVERY; Madison, WI, USA
ABSTRACT
EXTRACTMAN® improved isolation speed nine-fold and produced equivalent or better yields of purified DNA compared to tube-based isolation methods. EXTRACTMAN is versatile and compatible with a variety of existing paramagnetic bead-based isolation kits. Four DNA samples from two different organisms (mammalian and plant) were processed in parallel with no cross-contamination.
INTRODUCTION
DNA sequence analysis is an essential method in both research and diagnostics with applications in a wide range of scientific fields, including agricultural, biotechnology, food safety, forensics, and virology. In medical research and diagnostics, human DNA is evaluated for the presence of factors, such as the presence of viral DNA or mutations in cancer, which can affect the development and progression of disease. Effective and efficient sequence detection requires preparation methods that isolate and purify DNA from crude lysates and other complex biological samples.
"EXTRACTMAN achieved four DNA isolations nine times faster than the tube-based method."
In this application note, EXTRACTMAN (Figure 1) was used to quickly, easily, and efficiently isolate DNA from crude preparations of mammalian and plant cells.
Technology Overview
The technology makes use of paramagnetic beads, also referred to as paramagnetic particles (PMP), with surfaces that bind specific analytes. Selective binding enables exclusion-based sample preparation (ESP™), a class of simple and fast purification techniques in which analyte bound to paramagnetic beads is pulled through a liquid interface into a second phase (air or liquid).
Because ESP uses surface tension to gently and efficiently exclude unbound material from being transferred to the next phase, even weakly-bound species can be isolated.
EXTRACTMAN Mechanism and Workflow
EXTRACTMAN employs novel techniques that enable ESP, offering a fundamentally new process of analyte purification (Figure 2). With EXTRACTMAN, paramagnetic beads bound to the analyte are uniquely manipulated through the bind, wash, elute workflow using two opposing sets of magnets: (1) free-floating capture magnets housed in the sliding handle, and (2) manually actuated release magnets, which slide below the microplate and have opposing polarity to the capture magnets. Sample preparation proceeds in two steps. The first step involves paramagnetic bead capture onto the handle's bead capture strip followed by sliding the handle to advance the bead capture strip (and associated paramagnetic beads) from a first column of wells to a second column. The release magnet, initially positioned under the second column of wells, repels the capture magnets and attracts the paramagnetic beads from bead capture strip into the second column of wells. In the second step, the release magnets are advanced to a successive column of wells. As a result, the capture magnets are no longer repelled by the release magnets, and fall back to the lower position to recapture the paramagnetic beads. Repeating these two steps across the microplate carries the paramagnetic beads bound to analyte from the input, through the washes, and to the output (Figure 3, on page 2).
"...Gilson EXTRACTMAN® was used to quickly, easily, and efficiently isolate DNA..."
The process of capture-slide-release-slide allows the user to quickly perform four isolations in parallel. The simple sliding movements virtually eliminate user-to-user variability, whereas analyte recovery and purity in other existing manual techniques depend greatly on the user's skill in the art of performing careful aspirations. Additionally, the simplicity of this ESP™ method has lent itself to straightforward automation on Gilson PIPETMAX®. Thus, the advantages of EXTRACTMAN® are easily scalable to higher throughput.
This application note describes DNA isolation from cell culture lysate by three commercial kits that were simply and easily adapted for use with EXTRACTMAN. EXTRACTMAN achieved four DNA isolations nine times faster than the tube-based protocol (Figure 4). Translation of these commercial kits to EXTRACTMAN resulted in improved or equivalent yield while maintaining sample purity and integrity.
[Bar chart illustrating the time comparison for DNA isolation between EXTRACTMAN and standard tube-based methods, showing EXTRACTMAN is approximately nine times faster.]
MATERIALS AND METHODS
Cell Culture
eGFP-expressing MCF7 breast cancer cells (a generous gift of Dr. Elaine Alarid, UW-Madison, Wisconsin) were maintained in a humidified chamber at 37°C and 5% CO2. Culture medium consisted of DMEM with 10% FBS and 1% penicillin-streptomycin. Cells were passaged and harvested using 0.25% trypsin-EDTA.
Kit Adaptation
Three kits were evaluated: Agencourt® DNAdvance™ (Beckman Coulter; Brea, CA); Dynabeads® DNA DIRECT™ Universal Kit (Life Technologies; Carlsbad, CA); Magnesil® Paramagnetic Particles (Promega; Madison, WI) with Buffer RLT (QIAGEN; Germantown, MD). Tube-based DNA isolations were performed with a 6-Tube Magnetic Separation Rack (New England BioLabs; Ipswich, MA). Kits were used according to manufacturers' protocols with the following modifications: DNA was isolated from 104 cells for all kits, final volume for elution was 100 µL for all kits, cell culture samples were used in place of tissue, lysis/digestion was shortened to 1 hour for AGENCOURT DNAdvance kit. For EXTRACTMAN isolations the DNAdvance and Dynabeads DNA DIRECT Universal Kit were further modified: sample lysate input volume was 420 µL for all kits (maintaining 104 cells/isolation and paramagnetic bead number was the same as the tube-based equivalent), all wash and elution volumes were 100 µL.
EXTRACTMAN Device Operation
Paramagnetic bead-bound DNA was captured in the input and transferred to the wash wells by sliding the handle with the paramagnetic beads held in place on the bead capture strip by the capture magnets. The paramagnetic beads were released into the wash wells and re-captured by moving the release magnet under and away from the current wash well respectively. This wash sequence was repeated for the appropriate number of wash columns. After re-capture in the final wash, DNA adsorbed to the paramagnetic beads was moved to the elution buffer output by again sliding the handle. The paramagnetic beads were released in the output well using the release magnet.
RESULTS
Yield Quantification
qPCR was used for relative quantification of DNA yield and as an indicator of sample integrity for downstream assays. Quantification was verified using the Qubit® 2.0 Fluorometer and dsDNA HS Assay Kit (Invitrogen; Carlsbad, CA).
Input Contaminant Exclusion from Output
Nonspecific carryover of contaminants from the input to the wash and elution wells was measured by spiking tartrazine (Sigma-Aldrich, St. Louis, MO) into the sample input. A CLARIOstar® microplate reader (BMG LABTECH; Ortenberg, Germany) was used to generate standard curves and quantitate the fraction of contaminant tartrazine from the input transferred to subsequent wells.
More than 99% of Starting Contaminants Excluded from EXTRACTMAN Output: Tartrazine, a food dye with a broad linear range of absorbance at a wavelength of 450 nm, was used as a reporter contaminant to assess and quantify the extent to which nonspecific carryover might contribute to contamination of the purified sample. Absorbance values for the elution well indicate that more than 99% of the input tartrazine was eliminated.
[Bar chart showing Qubit yield quantification for DNA isolated from MCF7-GFP cells using EXTRACTMAN vs. tube-based protocols for three different kits. Below, a similar bar chart shows qPCR yield quantification.]
Maintenance of Sample Integrity
The potential for cross-contamination was tested by loading an EXTRACTMAN microplate with MCF7 and cornhusk lysate, alternating mammalian and plant samples in the four rows A-D in the EXTRACTMAN microplate. After DNA isolation all samples were tested for mammalian and plant DNA by PCR using prime/probe sets for GAPDH and corn alcohol dehydrogenase (ADH) respectively (Applied Biosystems; Carlsbad, CA).
[Bar chart showing the percentage of contaminant elimination for different DNA isolation kits. To the right, a table indicates species detection (Human DNA, Corn DNA) in the sample input versus the detected output for four different lanes, demonstrating no cross-contamination.]
SUMMARY
- EXTRACTMAN® utilizes the simple and robust ESP™ technique and its operation is rapid, efficient, and straightforward—just slide the handle to bind, wash, and elute.
- The bead capture strips enable high yield purification of multiple samples in parallel with no cross-contamination, loss in yield, or compromise in sample purity and integrity.
- EXTRACTMAN technology is flexible, and is compatible with a variety of magnetic-based kits for isolation of DNA, proteins, and other analytes.
REFERENCES
- Berry SM, Chin EN, Jackson SS, Strotman LN, Goel M, Thompson NE, Alexander CM, Miyamoto S, Burgess RR, Beebe DJ. Weak protein-protein interactions revealed by immiscible filtration assisted by surface tension. Anal. Biochem. 15:447:133-40.
- Casavant BP, Guckenberger DJ, Beebe DJ, Berry SM. Efficient sample preparation from complex biological samples using a sliding lid for immobilized droplet extractions. Anal. Chem. 86: 6355-62.
- Rahman MM, Elaissari A. Nucleic acid sample preparation for in vitro molecular diagnosis: from conventional techniques to biotechnology. Drug Disc. Today. 17: 1199-207.
Trademarks
All product and company names are trademarks™ or registered® trademarks of their respective holders. Use of the trademark(s) in this document does not imply any affiliation with or endorsements by the trademark holder(s).
- Agencourt® and DNAdvance™ (Beckman Coulter)
- Exclusion-based Sample Preparation (ESP™) (Salus Discovery)
- Dynabeads® and DNA DIRECT™ (Life Technologies)
- Magnesil® (Promega)
- Qubit® (Invitrogen)
- CLARIOstar® (BMG LABTECH)
Contact Information
Gilson, Inc.
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Tel: 608-836-1551 or 800-445-7661 F 608-831-4451
Gilson S.A.S.
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