Analytical and Bioanalytical Chemistry
Electronic Supplementary Material
D. Kalyanasundaram · J.-H. Kim · W.-H. Yeo · K. Oh · K.-H. Lee · M.-H. Kim · S.-M.
Ryew · S.-G. Ahn · D. Gao · G. A. Cangelosi · J.-H. Chung
1. Gel electrophoresis of the captured λ DNA
The aims of the experiment are to study (1) the effect of an electric field on DNA concentration and (2) the integrity of the released DNA from the microtip. For characterization, λ DNA and human genomic DNA from saliva samples were used. For comparison with a commercial kit, the same samples were processed by a DNA sample preparation kit (Qiagen® QIAmp DNA mini kit).
For gel analysis, λ-DNA was purchased from New England Biolabs. The concentration of λ DNA was 500 μg/mL. For capture of λ DNA, 5 μL solution was loaded on to a metallic ring. To study the damage of DNA due to the microtip device, only λ DNA spiked in 1X TE buffer were used without any reagents. As illustrated in the protocol
(Fig. 2), the volume of P-K and SDS was only 4 µL for 100 µL saliva samples and hence not used. An AC electric field of 20 V peak to peak (V pp
) at 5 MHz was used. The immersion time of the chip in to the ring was 30 seconds. After DNA capture, DNA was eluted by heating the microchips in a vial with 30 μL of 1X TE buffer of 8.5 pH. The eluted DNA solution of 10 μL was mixed with 1 μL of dye and loaded in to the wells of the agarose gel.
Agarose SeaKEM gold from Lonza technologies (Walkerswille, MD) was used for the gel electrophoresis. The gel analysis was conducted only for λ DNA due to the high concentration. The concentration of the agarose in the gel was 0.8 percent. 1X TAE
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buffer was used for the experiments. Mono cut λ-DNA from New England biolabs was used as the ladder. The electrophoresis run at 20 V for 16 hours and was post-stained for one hour before imaging. Real time PCR (qPCR) of the same samples was also conducted for comparison. The primers for λ-DNA experiments were 5’ – GAT GAG
TTC GTG TCC GTA CAA CTG G-3’ (25 bases) and 5’- GGT TAT CGA AAT CAG
CCA CAG CGC C-3’ (25 bases)
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.
The results for the gel electrophoresis and the qPCR analysis are shown in Fig S1. For λ
DNA, the recovery with an electric field (lane 3) shows higher fluorescence intensity than that without an electric field (lane 2). The band distribution of the captured DNA with the microtip device is very similar to that of the 1/10 th
diluted λ DNA. The λ DNA processed with the commercial kit has lower fluorescence intensity in the band but the band spread toward a smaller length of DNA.
(a) (b)
Fig. S1.
(a): Gel electrophoresis image of λ DNA recovered by a microtip device without an electric field (lane 2), with an electric field (lane 3), DNA by the commercial kit (lane
4) and 1/10 th
of original λ DNA sample (lane 5). Mono-cut λ DNA was used as the ladder
(lane 1). The unit of the numbers in the ladder is kbp. (b): Threshold cycles of qPCR analysis for λ DNA and saliva samples (n=3). The error bar represents standard deviation.
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According to the results, the electric field does not appear to damage λ-DNA in the recovery process. The silica micropore-based kit evidently damages λ-DNA in the purification step. The corresponding qPCR results are shown in Fig. S1(b). The λ-DNA without an electric field showed a threshold cycle of 11, which was lagged by 7 cycles in comparison with the λ-DNA with an electric field. It was surprisingly found that the λ-
DNA recovered by the microtip device with an electric field and the commercial kit showed equivalent threshold cycles. Since the length of the amplicon was 100 bps, the damage of λ-DNA could not be expressed in qPCR analysis.
For saliva samples, the threshold cycles for microchips without and with an electric field shows 2 cycle difference [Fig. S1(b)]. However, the threshold cycles of the microchips without an electric field show a larger error bar.
According to the results, the electrical conductivity of the solution and the presence of charged contaminants can affect the capturing and the PCR amplification. In pure λ-DNA solutions where the conductivity is low, an electric field plays a significant role in concentrating the DNA. However, the difference is reduced when the complex solution of saliva is used. The results of the microtip device with an electric field are comparable with those of the commercial kit. However, DNA was damaged with a higher degree for the commercial kit.
2. qPCR analysis and gene information
For qPCR, 5 μL of the eluted solution was used. Sybr GREEN Express® (Invitrogen,
Carlsbad, CA, USA) mastermix was used as the fluorescence dye. For qPCR analysis, 14
μL of master mix, 3 μL of each of the forward and reverse primers (2 μM), and 5 μL of eluted DNA in buffer were used. The qPCR equipment was CFX 96 from BioRAD
(Hercules, CA).
In case of the buccal swab samples, the β actin gene was detected for nuclear DNA of human cells. Two different lengths (100 and 1500 bp) of PCR amplicons in the β-actin gene region were used to evaluate the DNA length for the microchips and the commercial kit. The primer sequences for 100 bp amplicon were: forward primer: 5' -ACC CAC ACT
GTG CCC ATC TAC-3' (21 bases) and reverse primer: 5' - TCG GTG AGG ATC TTC
ATG AGG TA - 3' (23 bases)
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. For a longer segment of DNA, a 1500 bp amplicon was
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used. The primers were: 5’-CAA CCT GTC CTC TGC CTC TGC C-3’ (22 bases) and
5’-TCC CAC TTG CAG ACG CTC CCA –3’ (21 bases). The primers for 1500 bp amplicon of β-globin were designed by the NIH’s primer design tool 3
.
In case of the saliva samples, both nuclear and mitochondrial genes were detected in the comparison of the microchip and the commercial kit. For nuclear genes, the same amplicon of 100 bp was used as that of the buccal swab samples. Mitochondrial DNA was detected by amplifying the cytochrome c oxidase gene. The primers were (a) forward primer: 5'-TCG CCG ACC GTT GAC TAT TCT CT-3' (23 bases) and (b) reverse primer: 5'-AAG ATT ATT ACA AAT GCA TGG GC-3' (23 bases) 4 .
3. AFM study
Atomic force microscopy (AFM) measurement was performed to understand and assess the intactness of DNA sample preparation. For the AFM study, human cells at concentration of 10
6
cells/mL were used. The John Stam lab at Genome Science
Department at University of Washington kindly offered K562 leukemia cells (ATCC,
Manassas, VA). The cells were cultured in RPMI medium 1640 (Invitrogen, Carlsbad,
CA, USA) supplemented with 10% FBS, 2 mM L-Glutamine, 1 mM pyruvate and penicillin/streptomycin. The cells were harvested at a density of 10
7
cells/mL. The Cells were counted by hymacytometer (Hausser Scientific, Horsham, PA, USA). The cells were diluted to 10 5 cells/mL and processed by the buccal swab protocol in Fig. 2.
For high-resolution AFM imaging, an ultra-sharp, high resolution AFM tip (tip radius: <
2 nm, type: MSS_NCHR_13, NanoAndMore USA, Lady’s Island, SC) was used
(Dimension 3100 SPM, NanoTech User Facility, University of Washington). The scan rate was 1 Hz in a tapping-air mode.
To characterize DNA and proteins, the molecules were immobilized on freshly cleaved mica (Ted Pella, Inc., Redding, CA). The disk-shaped mica substrate was coated with
Poly-L-lysine (PLL; Sigma-Aldrich, St. Louis, MO). 10 μL of PLL solution was dropped on the mica, and incubated for 1 minute. The mica was rinsed in pure water three times, and dried with nitrogen. Then, 5 μL of sample solution was placed on the PLL-coated mica surface for 5 minutes. To dry the sample completely, the mica substrate was placed
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under the laminar flow bench (Terra Universal, Model: 1686-54B, Fullerton, CA) for 10 minutes before AFM study.
The cells (10
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cells/mL in the volume of 100μL) were lysed using P-K and SDS according to the buccal swab protocol shown in Fig. 2. The cell suspension was centrifuged at 500 g for 10 minutes. The supernatant of 80 μL was discarded, and 20 μL was used for the experiment. 1 μL P-K and 4 μL of SDS (0.28% concentration) were added the cell solution. The mixture was then heated at 60 °C for 10 minutes for cell lysis. Similarly, 5 μL of cell lysis solution was loaded in a metallic coil for DNA capture.
5 MHz AC signal of 20 Vpp was applied between the electrodes. The chips containing the microtips were immersed in to the cell lysate for 30 seconds. For elution of the DNA, the microchips were heated at 70 °C for 3 minutes in a sealed vial filled with 30 μL TE
8.5 pH buffer.
Fig. S2.
(a) AFM image of DNA processed by the commercial kit (b) AFM image of
DNA eluted from a microtip device. Inset shows a magnified view of DNA and protein molecules. (c) Height profile of protein molecules and DNA
Fig. S2 shows the AFM image of the DNA processed with the commercial kit and the microtip device. The DNA sample of the commercial kit has high concentration of ions
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and protein molecules, which makes the images blurred. The DNA from the microtip device was clearly imaged. The bright spots on the images represent protein molecules
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.
A continuous strand of 3 μm-long DNA can be observed for the DNA processed by the microtip device. The height profile shows that the long strand is a DNA molecule.
4. Storage test on K562 cells
Fig. S3. Storage test of genomic DNA of K562 leukemia cells on microchips for 6 months
To test the storage functionality of the tip, K562 leukemia cells were used. The cells were processed by the buccal swab protocol shown in Fig. 2. After DNA capture, the chips were stored in a clean vial at room temperature and were eluted at different time periods from the time of capture. In the storage, dessicants or chemicals were not used in the vial.
Sets of three chips were eluted after 1 day, 2 days. 7 days, 14 days, 30 days, 90 days and
180 days. Total 21 individual chips were prepared. For elution of the DNA, the microchips were heated at 70 °C for 3 minutes in a sealed vial filled with 30 μL TE 8.5 pH buffer. The eluted DNA was analyzed by qPCR. Fig. S3 shows the consistent threshold cycles of 23.5 with a standard deviation of 0.5 cycles. The results show that human genomic DNA prepared by the buccal swab protocol can be preserved at room temperature for 6 months.
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