ON-CHIP DEVICE FOR ISOTHERMAL,
CHEMICAL CYCLING
POLYMERASE CHAIN REACTION
Alexandre Persat1 and Juan G. Santiago1
1
Stanford University,USA
ABSTRACT
We demonstrate a novel chemical cycling polymerase chain reaction (ccPCR)
technique for DNA amplification in a fully working device where temperature is
held constant in space and time. We demonstrate successful ccPCR amplification
while simultaneously focusing products via isotachophoresis (ITP) for identification
of the environmental bacteria E. Coli. We electrophoretically drive the DNA sample
with ITP through a series of high denaturant concentration zones. The denaturant is
neutral so the DNA experiences alternatively low and high concentrations. This effectively replaces the thermal cycling of classical PCR (Figure 1). We performed
ccPCR with end-point detection and real-time fluorescence monitoring.
KEYWORDS: Polymerase chain reaction, isotachophoresis, isothermal DNA amplification
Figure 1: Conceptual representation of ccPCR. Zones of high
denaturant concentration flow
in opposite direction of DNA
template electromigration. The
template experiences a chemical cycling that mimics the
thermal cycling of classical
PCR.
INTRODUCTION
PCR is the platform of choice for biological and medical assays. Other isothermal nucleic acid amplification methods have not successfully competed with PCR.
Techniques such as the nucleic acid sequence based amplification (NASBA) [1] or
the rolling-circle amplification [2] have more complex chemistries and lack the versatility of PCR. Microfluidic platforms employing traditional PCR have competed
for faster thermal cycling [3], miniaturization [4], and sensitivity [5]. We previously
described and demonstrated preliminary experiments towards ccPCR [6]. In this
paper, we present a fully functional device including simultaneous flow control and
isotachophoretic focusing, and successful isothermal amplification with end-point
and real time detection. ccPCR has potential for simpler chemistry and device fabrication, enhanced replication fidelity, faster amplification, more accurate quantitation
and lower power consumption than traditional PCR.
EXPERIMENTAL
We leverage the dependence on solvent type and concentration on DNA melting
temperature Tm. Tm decreases with formamide and urea concentrations (Figure 2),
Twelfth International Conference on Miniaturized Systems for Chemistry and Life Sciences
October 12 - 16, 2008, San Diego, California, USA
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solvents of choice in denaturing gel electrophoresis [7]. At isothermal conditions,
high denaturant concentration initiates melting of DNA. We focus DNA with ITP
and control flow to create spatiotemporal variations of denaturant concentration.
DNA electromigrates against the flow and is held stationary upon balance of adevtion and electromigration velocities. The neutral denaturant clouds flow through the
focused polynucleotide zone (Figure 3). ITP focusing is robust and protects DNA
from dispersion.
Figure 2. Measured effect of formamide
and urea concentration on melting temperature Tm of the 16S rRNA gene from
E. Coli. Tm decreases with increasing
urea and formamide concentrations.
Figure 3. Schematic of the on-chip
ccPCR. DNA template (green) focuses
between with ITP. Upon balance of flow
and electromigration velocities, DNA is
stationary. A flow control scheme at the
cross creates discrete pulses of denaturant. a) DNA experiences a high denaturant concentration and denatures. b)
DNA anneals a primer which is then extended by a polymerase.
We introduce Taq polymerase, primers, dNTPs and magnesium in the leading
electrolyte (LE) (Tris hydrochloride, potassium chloride) and use Tris HEPES as
trailing electrolyte (TE). We use a 194 bp fragment of the rRNA gene from E. Coli
as template and initially focused 104 to 106 copies between LE and TE. We monitor
the reaction by SYTO 13 fluorescence and use an electrophoretic spacer between the
primers from the PCR product (Figure 4). Working temperature is 55ºC to ensure
specific annealing, and denaturant is 40% formamide 4 M urea. We control flow in
a borosilicate microchannel with an off-chip flow sequencer. Polyvinylpyrrolidone
coating limits polymerase adsorption on glass.
Figure 4. ITP of DNA in ccPCR conditions. A non fluorescent spacer (here benzoate) separates DNA and primers, reducing allowing PCR product localization.
RESULTS AND DISCUSSION
We performed end-point detection after successful amplification by ccPCR (Figure 5a). Initially, the fluorescent signal is below the limit of detection. After 40 cycles, the electropherogram shows a peak corresponding to ccPCR product. We present a sample result of real time ccPCR on Figure 5b, where we measure
fluorescence at the end of each cycle. For that particular run, fluorescence increases
above background at cycle 18.
Twelfth International Conference on Miniaturized Systems for Chemistry and Life Sciences
October 12 - 16, 2008, San Diego, California, USA
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Figure 5. End point detection and real-time monitoring of ccPCR. a) Isotachopherograms of PCR product zone before (left) and after 40 ccPCR cycles (right).
This type of end point detection allows identification of DNA sequence of interest.
b) Real time fluorescence monitoring of ccPCR. Initially, the PCR product fluorescence signal is below limit of detection.
CONCLUSIONS
We successfully demonstrated a novel chemical cycling polymerase chain reaction (ccPCR) technique for DNA. We successfully identified environmental bacteria
E. Coli by ccPCR amplification with simultaneous separation and focusing of product. This is the first time DNA amplification by PCR has been performed isothermally. Performing the reaction at lower, constant temperature relaxes the requirement for thermostable polymerase. The ccPCR combines electric field driven
migration of the sample and counterflow of reactants in a microchannel, which to
our knowledge has never been described. Current work involves accurate DNA
quantitation and integration of reverse transcription for RNA quantitation.
ACKNOWLEDGEMENTS
The authors would like to acknowledge Tomoyuki Morita for his contribution at
the early stages of this work, and Ebara Research Corp., Ltd.
REFERENCES
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[3] Giordano, B.C., et al., Polymerase chain reaction in polymeric microchips: DNA
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[6] Persat, A., Morita, T., and Santiago J.G. Towards on-chip isothermal polymerase
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