Isolation and purification of biomolecules (single- and double- stranded DNA, proteins, mRNAs, miRNA, total RNAs), as well as specific organs and cells facilitate a broad range of downstream applications including cloning, gene and protein expression studies, protein-protein interactions, transfection, clinical diagnostics, immunology, PCR and qPCR, cDNA library synthesis, Sanger and NGS sequencing.
Magnetic separation is simple and has good recovery efficiency and purity. It is used in this bio-separation arena and has gradually replaced the traditional liquid phase and solid phase separation methods, which need extensive configuration or vacuum filtration process that are hard to automate.
However, when reproducibility is critical for obtaining results, manual performance varies significantly between operators and even for the same operator. Procedures can be standardized by automation, which reduces this variability significantly. However, automation may be limited in other aspects, for example efficient bead collection or liquid evacuation. The BeadTender method (Figure 1), is an automated solution for magnetic bead manipulation using the Andrew robot.
Figure 1: In-tip separation of beads from samples by the BeadTender method and the Andrew robot.
It comprises a specific manipulation and magnetic design which enables bead separation, washing and elution, all inside a conventional pipette tip. The proprietary method (patents pending) improves the reproducibility of the purified biomolecules and has the highest efficiency and flexibility in the sample and elution volumes.
The entire BeadTender method takes place within a conventional tip connected to a conventional pipette. The process encompasses aspiration of sample, active mixing of bead, bead washing, bead pelleting, incubation, drying, and sample elution. Therefore, users are spared completely from manual operations and the repetitive strain injury caused by the manual resuspension of the bead at various steps.
The BeadTender is entirely independent of the consumables holding the sample and is scalable for all common volume ranges of biological applications. The parameters for bead manipulation are adjustable and optimizable and can be adapted to any bead type or protocol. This means that the BeadTender offers a flexible and universal solution to the small-scale automation of magnetic bead manipulation.
The BeadTender Method Improves the Reproducibility of DNA Purification
Two procedures were used to test the performance of the BeadTender method: (1) genomic DNA extraction from 250 µL human saliva samples contained in 2 mL microcentrifuge tubes, and (2) purification of 50 µL PCR products contained in 0.2 mL PCR tubes. The sample of saliva was treated at 50oC overnight and was divided into eight aliquots of 250 µL. The KAPA HiFi HotStart ReadyMix PCR Kit (KAPA) was used to generate the 700 bp PCR products.
Numerous PCR reactions were pooled and then split into 10 samples of 50 µL. A pipetting robot Andrew model 1000G was used to perform five replicates of PCR purification and four replicates of saliva genomic DNA extraction. For PCR purification, the 250 µL tips from Biotix were used with the P200 pipette. For genomic DNA extraction, 1000 µL Diamond tips (Glison D1000) with the P1000 pipette, were used. An experienced human operator carried out the same number of replicates, performing the process using the same material.
For purification, Andrew mixed 90 µL of AMPure XP magnetic beads (Beckman Coulter), with 50 µL PCR samples. The bead pellets were washed twice with 140 µL 80% EtOH, and the tips holding the bead pellets were washed once using 50 µL water. The purified PCR products were then eluted in 50 µL 10 mM Tris buffer (pH 8).
Using reagents from the Mag-Bind Blood and Tissue DNA HDQ Kit (Omega Biotek), the saliva genomic DNA was extracted. Each sample was mixed with 290 µL of lysis buffer AL and 20 µL of Proteinase K (20 mg/mL). The samples were then incubated at room temperature for five minutes and mixed by pipetting up and down 200 times with 20 µL HDQ magnetic beads and 400 µL HDQ binding buffer.
Next, the bead pellet was washed twice with the VHB buffer, once with the SPM buffer, and once with water, all in 980 µL. The sample was then air dried for five minutes and resuspended in 200 µL of elution buffer to elute DNA. For manual pelleting, the 96S magnetic plate (Alpaqua) and the Chemagic Stand 2x12 (Perkin Elmer) were used as a magnetic separator. The manual bead resuspension steps were all done by vortexing for 60 seconds, except for the final step with the elution buffer where manual pipetting up and down 50 times was used.
All resuspension and pelleting steps for the automatic procedure took place inside the BeadTender by Andrew. With the manual method, samples were processed simultaneously, whereas the BeadTender samples were processed sequentially. Manual operation, including protocol execution, took 38 and 35 minutes for the four saliva DNA extractions and five PCR clean ups, respectively. Using the Wizard of Andrew Lab V1.5 (under release) to design protocols and preparing samples for Andrew and BeadTender these processes took seven and five minutes, respectively.
The purified DNA samples were diluted 50x or 100x so they could be quantified using the Qubit HS DNA assay (Thermo Fisher Scientific). Compared to those of the experienced human operator, the coefficient of variations (CV) from the DNA yields from saliva and PCR product performed by Andrew and BeadTender are eight times and three times lower. This indicates a significant improvement with regards to reproducibility (Figure 2).
Figure 2: Reproducibility of DNA extraction by BeadTender vs. human operator.
Two downstream applications were used to evaluate the purity of the DNA samples extracted manually and by the BeadTender. To evaluate the cleaned-up PCR products, 4 µL was sequenced by the Sanger sequencing method. A 50 µL PCR reaction was performed with 2 µL template DNA and the primers for human gene Hemoglobin Beta for the evaluation of the saliva genomic DNA. From all eight genomic DNA samples, an amplicon of the expected size, approximately 900 bp, was successfully amplified (Figure 3A). All Sanger-sequenced cleaned-up PCR products showed clean chromatograms with single sharp peaks for all 700 nucleotide positions (Figure 3B).
Figure 3: Purity of genomic DNA (A) and cleaned-up PCR products (B) extracted by BeadTender and manually.
These results show that all contaminants (unincorporated nucleotides and primers, PCR inhibitors, left-over salt and enzymes, by-product primer dimers) are removed by the BeadTender method with excellent efficiency.
BeadTender – Consumable – Independent Magnetic Separation
The workflow of magnetic separation by BeadTender is entirely contained within a pipet tip. Depending on the applications, this can accommodate samples as low as 5 µL and as high as 5 mL. The sample begins in any consumable of choice, where samples have been mixed with the appropriate magnetic bead type for capturing the biomolecules of interest (Figure 4A). The sample-bead mixture is aspirated into a pipet tip. This is then moved to a region where a suitably designed magnetic field can sweep and collect the beads into a pellet (Figure 4B).
Once the supernatant is separated physically from the beads by the combination of pipetting action and magnetic field, it is discarded and the beads are kept intact inside the tip. An ethanol mixture is then aspirated in the same tip and magnetic mixing is performed to improve the bead washing quality. Following this, the beads are pelleted (Figure 4C). The ethanol is then discarded and, without touching the bead pellet, water is aspirated into the tip to remove any remaining ethanol, and the pellet inside the tip is dried (Figure 4D).
Finally, the elution buffer of choice is drawn up inside the tip at the volume desired, and the biomolecules are released from the beads by resuspending the beads in the pellet using active magnetic mixing inside the BeadTender (Figure 4E). Having released the molecules of interest, the beads are now pelleted inside the tip and are separate from the elution buffer that contains the biomolecules (Figure 4F).
The eluted solution is transferred to a new destination consumable (Figure 4G) and then the tip containing the used beads is discarded. The entire process is carried out by the Andrew pipetting robot, without any user intervention. The operator only needs to supply the beads, samples, elution buffer of choice, wash buffers and clean consumables for the final purified products.
Figure 4: BeadTender design and working principle.
All procedure parameters are entirely adjustable in the graphical user interface of the software Andrew Lab, for example buffer volume, resuspension time and pelleting time. Therefore, the method can be fine-tuned to optimize purity and efficiency according to sample viscosity, type of bead, pH or ionic force of the buffer etc.
The BeadTender enables isolation and purification of biomolecules inside a conventional pipette tip. It offers flexibility of sample and elution volumes, independently of source and destination consumables. Compared to the performance of a skilled human operator, the reproducibility is improved by up to eight times. All of this is accomplished hands-off, by the Andrew robot.
About Andrew Alliance S.A.
Andrew Alliance is an independent, privately financed company, based in Geneva, Boston and Paris. The company was created in March 2011.
Andrew Alliance is dedicated to advance science by working with scientists to create a new class of easy-to-use robots and connected devices that take repeatability, performance, and efficiency of laboratory experiments to the level required by 21st-century biology.
Start with meeting customer needs, end with customer feedback.
Andrew Alliance delivers solutions that are focused on customer needs, both today and in the future. Our products are manufactured to the highest standards, using a range of carefully selected, proven, and sustainable technologies, that ensure both high performance and reliability. We actively seek continuous customer feedback, in order to guarantee the best possible design outcomes.
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