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Stanford University

Stanford Microfluidics Laboratory

On-chip Device for Isothermal, Chemical Cycling Polymerase Chain Reaction (ccPCR)

Principal Investigators: J.G. Santiago and Alexandre Persat


The polymerase chain reaction (PCR) is a ubiquitous platform in biological and medical assays.  PCR can amplify a specific DNA sequence with high sensitivity (single copy).  In particular, it is commonly used for identification of DNA, RNA (with initial reverse transcription, RTPCR) and also quantification (quantitative PCR, qPCR) of nucleic acids.  Examples of applications are: diagnosis of hereditary disease, forensics, gene expression profiling, pathogen detection, etc.

 

In classical PCR, double stranded DNA (dsDNA) experiences a three-step thermal cycle illustrated in figure 1.  The first step, denaturation at ~ 95°C, disrupts all hydrogen bonds between the two strands of the template, creating two complementary free strands of DNA.  The second step, annealing at ~ 55°C, allows specific binding of primers to the parent single-stranded DNA (ssDNA). Primers are specifically designed short ssDNA that are complementary to the 5’ ends flanking the sequence of interest.  Finally, in the extension step at ~ 72°C, a DNA polymerase binds to the priming sites and elongates the primer.  Ideally, after each cycle, the number of DNA copies in the reactor doubles.

 

Figure 1: Schematic of the classical PCR.  The double stranded DNA template experiences three steps temperature cycles where it denatures, anneals a primers and is extended by a polymerase.  In ideal conditions, the number of copies doubles after each cycle, and increases by 2N folds after N cycles.

 

We have demonstrated a novel chemical cycling polymerase chain reaction (ccPCR) technique for DNA amplification 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 (figure 2 and 3).  The denaturant is neutral so the DNA experiences alternatively low and high concentrations (movie).  This effectively replaces the thermal cycling of classical PCR.  We performed ccPCR with end-point detection and real-time fluorescence monitoring (figure 4).  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 and significantly reduces power consumption of the instrument.  Current work involves accurate DNA quantitation and integration of reverse transcription for RNA quantitation.
           

Figure 2: 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.

 

Movie 1: Experimental demonstration of chemical cycling.  We focus a fluorescent analyte with ITP (here fluorescein, green, represents DNA) and balance electromigration and bulk flow velocities to maintain the sample stationary in the microchannel.  The analyte experiences successively high and low concentrations of neutral specie (here rhodamine B, red, represents denaturant). 

 

Figure 3: Schematic of the ccPCR.  (a) DNA template (green) is initially injected between LE and TE.  (b)Upon application of an electric field, DNA focuses between TE and LE with ITP.  Upon balance of flow and electromigration velocities, DNA is stationary in the microchannel.  A precise flow control scheme at the cross creates discrete pulses of denaturant.  (c) DNA experiences a high denaturant concentration and denatures.  (d) DNA anneals a primer which is then extended by a polymerase.

 

Figure 4:  Experimental results of the 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.  Here, the fluorescent signal rises above background at cycle 18.

See related publications here