(320b) Simultaneous Purification and Fractionation of Nucleic Acids and Proteins From Complex Samples Using Isotachophoresis

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SIMULTANEOUS
PURIFICATION AND FRACTIONATION OF NUCLEIC ACIDS AND PROTEINS FROM COMPLEX
SAMPLES USING ISOTACHOPHORESIS

Yatian Qu, ¹ Lewis A.
Marshall, ² Juan G. Santiago¹

¹
Department of Mechanical Engineering, Stanford University

² Department
of Chemical Engineering, Stanford University

Stanford, CA 94305, USA

We report on the
development of a novel integrated sample preparation technique to simultaneously
extract nucleic acids and proteins from complex biological samples using
isotachophoresis (ITP). ITP is an electrophoretic technique that both separates and
pre-concentrates ions based on their electrophoretic mobility.
ADDIN EN.CITE
<EndNote><Cite><Author>Persat</Author><Year>2009</Year><RecNum>10</RecNum><DisplayText><style Marshall, L.A., Santiago,
J.G.</author></authors></contributors><titles><title>Purification
of Nucleic Acids from Whole Blood Using
Isotachophoresis</title><secondary-title>Analytical
Chemistry</secondary-title></titles><periodical><full-title>Analytical
Chemistry</full-title></periodical><pages>9507-9511</pages><volume>81</volume><number>22</number><dates><year>2009</year></dates><urls></urls></record></Cite></EndNote>¹ Our chip
contains a single input sample reservoir connected to two channels leading to
output reservoirs.  Anionic ITP in
one channel is used to extract and purify DNA and deliver it to the DNA output reservoir. Simultaneously,
the second channel uses cationic ITP to extract and purify proteins, preserving
their natural folding state. Purified DNA and proteins are thereby fractionated
and delivered to respective output reservoirs. The protein extraction can be
configured to exclude albumin, a highly abundant species with little analytical
value. The DNA is purified away from species that inhibit enzymatic
amplification such as polymerase chain reaction (PCR).

We
designed and fabricated the novel microfluidic device shown in Figure 1 to
demonstrate the technique. The device is fabricated from polydimethylsiloxane
(PDMS) and glass and includes two "C" shaped channels leading from a single sample
input reservoir at the center. The channels are 1 mm wide and 100 μm deep, with a volume of 4 μL for each branch. Each of two branches leading
form the center is about 40 mm long. The cationic ITP channel collects
positively charged protein species. The anionic ITP channel collects nucleic
acids, which are strongly negatively charged. Each branch has its own elution
reservoir, where the collected biomolecules can be pippetted
off the chip. Each elution reservoir is also connected to a buffering channel and
reservoir to provide additional pH-buffering capacity.

We successfully
demonstrated simultaneous extraction of extracellular DNA and proteins from 1 μL human serum samples. We optimized the buffer chemistry for this system by performing
numerical simulation using Stanford Public Release Electrophoretic Separation Solver
(SPRESSO), as shown in Figure 2. We visualized the process in a small version of
the separation device in order to obtain a series of images that capture the
dynamics of the ITP sample zone in both branches, and show these images in Figure
3. For purification experiments, we imaged focused nucleic acids zone (Figure 4) by using fluorescent dye TOTO-3. The focused protein zone (Figure 5) was monitored by
imaging spiked yellow fluorescent protein (YFP).

To
verify the purification of nucleic acids, we performed quantitative polymerase
chain reaction (qPCR) using primers for the human
gene BRAC2 (Figure 6). Even though human serum contains low abundance
of extra-cellular DNA,
ADDIN EN.CITE
<EndNote><Cite><Author>O&apos;Driscoll</Author><Year>2007</Year><RecNum>8</RecNum><DisplayText><style Article">17</ref-type><contributors><authors><author>Lorraine
O&apos;Driscoll</author></authors></contributors><titles><title>Extracellular
Nucleic Acids and their Potential as 
Diagnostic, Prognostic and Predictive
Biomarkers</title><secondary-title>Anticanser
Research</secondary-title></titles><periodical><full-title>Anticanser
Research</full-title></periodical><pages>1257-1266</pages><number>27</number><dates><year>2007</year></dates><urls></urls></record></Cite></EndNote>² ITP successfully recovered
sufficient material to detect this gene via qPCR. Unprocessed
serum shows no amplification in qPCR, as expected due
to the effects of PCR inhibitors present in serum such as immunoglobulin G (IgG). To analyze the extracted proteins, we performed sodium
dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) followed by
silver staining.  We compared
samples extracted via ITP with samples directly applied to the gel, as shown in
Figure 7.  We recovered a
wide variety of proteins from the original sample, and successfully excluded albumin.

Simultaneous extraction and purification of
nucleic acids and proteins from a single crude biological sample is important
for ensuring comparability between genomic and proteomic results, and to
conserve precious samples.
ADDIN EN.CITE <EndNote><Cite><Author>Butt</Author><Year>2007</Year><RecNum>3</RecNum><DisplayText><style Article">17</ref-type><contributors><authors><author>Butt,
R. H., Pfeifer, T.A., Delaney, A., Grigliatt, T.A., Tetzlaff, W.G., Coorssen,
J.R.</author></authors></contributors><titles><title>Enabling
Coupled Quantitative Genomics and Proteomics Analyses from Rat Spinal Cord
Samples</title><secondary-title>Molecular &amp; Cellular
Proteomics</secondary-title></titles><periodical><full-title>Molecular
&amp; Cellular Proteomics</full-title></periodical><pages>1574-1588</pages><number>6</number><dates><year>2007</year></dates><urls></urls><electronic-resource-num>10.1074/</electronic-resource-num></record></Cite></EndNote>³ To our knowledge, our technique is unique in offering availability
of automated and simultaneous nucleic acids and
protein extraction in microfluidics platforms.

Figure 1. Device
design for simultaneous purification of nucleic acids and proteins from serum
using simultaneous cationic and anionic ITP processes. Each reservoir
holds approximately 8 µL liquid. Each separation channel has a volume of 4 µL.

Figure 2. Spatiotemporal representation of numerical
simulation results. (Top) Ion concentrations: each ion is given its
own false color. Over time, the DNA and proteins focus into sharp counter
migrating bands. (Bottom) Conductivity map: low conductivity causes high electric field in the
TE zones; fast
ions quickly migrate out.

Figure 3. Experimental data plotted in a spatiotemporal plot
showing the dynamics of protein and DNA zones purified in our chip. Yellow
fluorescent protein and DNA labeled with SYBR Green I were imaged using a custom
fluorescence set up using a stereoscope. The DNA and protein zones are clearly
visible propagating away from the sample zone.

Figure 4. Fluorescent image of DNA labeled with TOTO-3 in the ITP
zone. DNA migrates toward the anode.

Figure 5. Fluorescent
image of YFP in the cationic ITP zone. YFP migrates toward the cathode.

Figure 6. qPCR
results: extracted nucleic acids were amplified using primers for a 201-bp
section of the BRAC2 gene in the human genome. The extracted DNA samples
amplified, while control samples of water and unprocessed serum did not. The
resulting amplicons had a melting temperature of
74°C, which matches the prediction from the Promega amplicon melting tool.
ADDIN EN.CITE
<EndNote><Cite><Author>Promega</Author><RecNum>9</RecNum><DisplayText><style Page">12</ref-type><contributors><authors><author>Promega</author></authors></contributors><titles></titles><dates></dates><urls><related-urls><url>http://www.promega.com/resources/tools/biomath-calculators/
</url></related-urls></urls></record></Cite><Cite><Author>Promega</Author><RecNum>9</RecNum><record><rec-number>9</rec-number><foreign-keys><key
app="EN"
db-id="xw220stpa9fwf6e25zsvxseksdx5wedtz9e2">9</key></foreign-keys><ref-type
name="Web
Page">12</ref-type><contributors><authors><author>Promega</author></authors></contributors><titles></titles><dates></dates><urls><related-urls><url>http://www.promega.com/resources/tools/biomath-calculators/
</url></related-urls></urls></record></Cite></EndNote>⁴

Figure 7. Silver-stained SDS-PAGE of original serum sample,
and proteins extracted using ITP. We recover at least 17 visible protein bands
over a range of molecular weights. 
Protein bands below 21.5 kDa are faint but
detectable. Albumin is the most concentrated band in the serum sample, but is
not visible in the selectively extracted sample.

REFERENCES

 ADDIN EN.REFLIST 1.  Persat,
A., Marshall, L.A., Santiago, J.G., Purification of Nucleic Acids from Whole
Blood Using Isotachophoresis. Analytical
Chemistry 2009, 81 (22), 9507-9511.

2.  O'Driscoll, L., Extracellular Nucleic
Acids and their Potential as 
Diagnostic, Prognostic and Predictive Biomarkers. Anticanser Research 2007,  (27), 1257-1266.

3.  Butt, R.H., Pfeifer, T.A., Delaney, A.,
Grigliatt, T.A., Tetzlaff, W.G., Coorssen, J.R., Enabling Coupled Quantitative
Genomics and Proteomics Analyses from Rat Spinal Cord Samples. Molecular & Cellular Proteomics 2007,  (6), 1574-1588.

4.  Promega http://www.promega.com/resources/tools/biomath-calculators/