Microfluidic gradient PCR (MG-PCR): a new method for microfluidic DNA amplification
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- Zhang, C. & Xing, D. Biomed Microdevices (2010) 12: 1. doi:10.1007/s10544-009-9352-2
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This study develops a new microfluidic DNA amplification strategy for executing parallel DNA amplification in the microfluidic gradient polymerase chain reaction (MG-PCR) device. The developed temperature gradient microfluidic system is generated by using an innovative fin design. The device mainly consists of modular thermally conductive copper flake which is attached onto a finned aluminum heat sink with a small fan. In our microfluidic temperature gradient prototype, a non-linear temperature gradient is produced along the gradient direction. On the copper flake of length 45 mm, width 40 mm and thickness 4 mm, the temperature gradient easily spans the range from 97 to 52°C. By making full use of the hot (90–97°C) and cold (60–70°C) regions on the temperature gradient device, the parallel, two-temperature MG-PCR amplification is feasible. As a demonstration, the MG-PCR from three parallel reactions of 112-bp Escherichia coli DNA fragment is performed in a continuous-flow format, in which the flow of the PCR reagent in the closed loop is induced by the buoyancy-driven nature convection. Although the prototype is not optimized, the MG-PCR amplification can be completed in less than 45 min. However, the MG-PCR thermocycler presented herein can be further scaled-down, and thus the amplification times and reagent consumption can be further reduced. In addition, the currently developed temperature gradient technology can be applied onto other continuous-flow MG-PCR systems or used for other analytical purposes such as parallel and combination measurements, and fluorescent melting curve analysis.
KeywordsMicrofluidic gradient PCR (MG-PCR)Microfluidic DNA amplificationContinuous-flowTemperature gradientNature convection
The polymerase chain reaction (PCR) technique represents a major advance in molecular biology. It has been applied to a diverse range of basic research and application fields, including medical diagnostics, infectious and hereditary disease detection, forensic science, and genomics. Under optimal conditions, the PCR is very efficient; microgram quantities can be synthesized from a single-molecule DNA template. Optimization of PCR involves testing a number of variables, one of which is the primer annealing temperature. Although good primers are chosen, i.e., those that do not form dimmers by annealing of their 3′-termini and that are specific to one sequence in template DNA, annealing temperature optimization is important for PCR performance (Rychlik et al. 1990; Wu et al. 1991). If the annealing temperature is too low, non-specific DNA fragments are amplified, causing the appearance of multiple bands on agarose gels. If it is too high, the yield of the desired product, and sometimes the product purity is decreased due to poor annealing of primers (Rychlik et al. 1990). Fortunately, commercial gradient PCR machines can generate a temperature gradient up to 12 different annealing temperatures, and they have been successfully used in many fields such as site-directed mutagenesis (Padmakumar and Varadarajan 2003), improvement of the efficiency and reliability of RT-PCR using tag-extended primers (Sybesma et al. 2001), and identification of Panax Species in the herbal medicine preparations (Shim et al. 2005). However, conventional gradient PCR machines tend to be expensive; make a gradient up to only 12 different annealing temperatures spanning a range of 20°C. Recently, Chang and Lee have developed a volumed gradient PCR method, in which a traditional PCR machine can be converted into a gradient PCR machine (Chang and Lee 2005). Although this strategy can make as many annealing temperatures as possible by adjusting reaction volume, the accuracy of annealing temperature largely depends on one of titered volumes that may vary among users. In addition, the reaction volume also affects the specificity of DNA amplification, as similar to the annealing temperature does. Importantly, conventional PCR cycles are performed in a metal block thermal cycler that usually takes 2–3 h for thirty cycles; most of the reaction time is spent on cooling and heating during the reaction process. In addition, these PCR machines are bulky and energy intensive, making it hard to bring PCR (including gradient PCR) to point-of-care (POC) applications.
In recent years, great effort has been spent in miniaturizing the PCR technique in order to increase reaction throughputs and small-scale integration level while simultaneously minimizing reagent consumption and/or reaction time (Zhang et al. 2006, 2007a; Zhang and Xing 2007). The design and operation of microfluidic PCR can be classified into two principal categories: stationary chamber-based PCR and dynamic flow-based PCR. Originally, chamber-based PCR is performed in a PCR solution-containing chamber that is cycled between different temperatures (Wilding et al. 1994). Subsequently, the array chambers on a single chip have been developed for high-throughput DNA amplification and analysis (Fan and Quake 2007; Morrison et al. 2006; Nagai et al. 2001). This chamber-based stationary approach can produce PCR systems with the smallest footprint and the highest integration, but complex control instrumentation is required to thermally cycle the PCR solution among the desired temperatures. In addition, the thermal mass associated with the heater, PCR chamber and solution usually limits the flexibility to change the PCR speed.
Alternatively, the dynamic flow-based PCR performs DNA amplification by using the ‘time-space conversion’ concept. The amplification reaction occurs as the DNA sample continuously flows through a microfluidic channel during each temperature cycle. The entire PCR period depends only on the sample flow rate and the time needed for the sample to reach a thermal equilibrium. In 1998, Kopp et al. reported a continuous-flow PCR chip using a microfluidic serpentine channel embedded within a glass substrate (Kopp et al. 1998). Since then, some researchers have continued to improve the operation of this original device (Zhang et al. 2006, 2007a; Zhang and Xing 2007). Importantly, continuous-flow PCR for high-throughput applifications has been developed in a serial (Chabert et al. 2006; Curcio and Roeraade 2003; Dorfman et al. 2005; Kiss et al. 2008; Li et al. 2009; Obeid et al. 2003; Park et al. 2003; Schaerli et al. 2009; Zhang et al. 2007b) or parallel (Sun et al. 2008) format. For serial high-throughput amplification, either the sample can be a droplet entrained in an immiscible solvent (Chabert et al. 2006; Curcio and Roeraade 2003; Dorfman et al. 2005; Kiss et al. 2008; Schaerli et al. 2009), or numerous aqueous sample plugs can be separated by plugs of air (Li et al. 2009; Obeid et al. 2003; Park et al. 2003; Zhang et al. 2007b). For example, Dorfman et al. examined continuous-flow PCR using droplets in an immiscible, fluorinated solvent flowing in Teflon capillaries, without any detectable contamination between neighboring droplets (Dorfman et al. 2005). Park et al. demonstrated a segmented-flow PCR of four different samples for amplifying different DNA fragments (500, 323, 497, and 267 bp), where the air gap/bromophenol blue buffer plug/air gap was interposed between each sample to discourage the carryover from one segment to the following one. In the parallel format, different samples are amplified in different continuous-flow reaction channels. For example, Sun et al. recently reported a multichannel closed-loop magnetically actuated microchip for high-throughput, continuous-flow PCR (Sun et al. 2008). Four different genes, the 172-bp 35 S-promoter sequence that is specific for the detection of genetic modifications in soybeans, the soybean control 118-bp lectin gene LE1 that is detectable in both transgenic and conventional soybeans, the 211-bp cryIA gene specific for the transgenic maize, and the 226-bp invertase maize control gene were amplified simultaneously in the four different channels of the microchip, and 25 cycles were completed within 13 min. However, all these high-throughput PCR amplification and analysis systems (including stationary array chamber-based PCR and dynamic flow-based PCR (serial and parallel)) performed PCR of different samples using the same annealing temperature. In general, performing PCR for various gene segment amplifications from different template samples and/or different primer pairs requires different annealing temperatures, and an optimized annealing temperature can give a more effective and specific DNA amplification. Therefore, in order to effectively perform high-throughput PCR amplifications by microfluidic PCR, the annealing temperatures among different PCR reactions should be closely controlled, just like the conventional gradient PCR machine does.
We present here a microfluidic gradient PCR (MG-PCR) method for performing parallel DNA amplifications with different annealing temperatures. This is accomplished by using an innovative temperature gradient microfluidic system. When high temperatures (for example 90–97°C) and low temperatures (for 52–68°C) on the developed planar thermal gradient device are used for PCR temperatures, the two-temperature MG-PCR can be achieved. Here, as a demonstration and proof of principle, three-parallel PCR reactions with various annealing/extension temperatures were tested using the same gene segment to be amplified, where the flow of the PCR reagent in the closed-loop PCR reaction was driven by the buoyancy-driven nature convection. As the research background of our study and potential applications of our microfluidic temperature gradient technique, the other temperature gradient devices for microfluidic DNA analysis also have been introduced.
2 Temperature gradient devices for microfluidic DNA analysis
Over the last several years, the development of the temperature gradient microfluidic devices has attracted great interest, and has witnessed steady advances. Early in 2003, Kajiyama et al. demonstrated a temperature gradient DNA microarray hybridization chip for genotyping of four different loci in two genes (Kajiyama et al. 2003). Silicon-based chips with discrete, independently temperature-controlled islands were developed and temperatures at each island could be set at different values to obtain optimal distinction between perfect matches and mismatches (Kajiyama et al. 2003). Traditionally, DNA melting analysis is a time-based process. A recent technique, spatial DNA melting analysis, can detect fluorescence as a function of position instead of time, by creating a characteristic gradient within the DNA sample. Performing spatial DNA melting analysis in a temperature gradient device was first demonstrated by Mao et al. (Mao et al. 2002a, b). They utilized a poly(dimethylsiloxane) (PDMS) (Mao et al. 2002b) or glass (Mao et al. 2002a) substrate containing multiple microchannels that were each heated to a unique temperature on a linear temperature gradient device. In order to detect single nucleotide polymorphisms (SNPs), the channels were first filled with a concentrated solution containing a DNA dye and double-stranded oligonucleotides (30-bp) (Mao et al. 2002a). A similar method was also demonstrated by Baaske et al. (Baaske et al. 2007), where an infrared laser was used to generate a radial temperature gradient distribution between 20 and 100°C within a small (10 nl) volume. The thermal denaturing of a concentrated sample containing a dye-labeled DNA hairpin (33-bp) was performed in a snapshot (50 ms). Different from these two prior studies, Crews et al. recently reported simultaneous continuous-flow PCR amplification and spatial melting curve analysis starting from the non-designed and non-synthesized DNA templates within a one-dimensional linear temperature gradient (Crews et al. 2008a, c, 2009). The denaturing of the PCR product during each cycle was monitored by imaging the continuous-flow PCR on the temperature gradient device. A similar demonstration was also performed by Kinahan et al. (Kinahan et al. 2009), who measured DNA melting temperatures from two plasmid fragments within a linear temperature gradient to study the effects of substrate thermal resistance (including flow velocity and ramp-rate) on continuous-flow microchannel melting curve analysis. It should be noted that some temperature gradient microfluidic devices have been incorporated onto the chip-based capillary electrophoresis systems to perform DNA mutation detection (Zhang et al. 2007c; Buch et al. 2004, 2005), and microfluidic temperature gradient focusing (Balss et al. 2004; Ross and Locascio 2002).
Recently, the temperature gradient technique has been used for the continuous-flow PCR applications. By using this technique, the original chip-based continuous-flow PCR device reported by Kopp et al. has been improved to a certain extent. For example, Crews et al. used a microfluidic serpentine channel embedded within a glass substrate, as similar to Kopp et al.’s work, and substituted a single steady-state temperature zone for three distinct temperature zones reported by Kopp et al.. As a result, the serpentine channel continuous-flow PCR chips on a temperature gradient device were performed (Crews et al. 2007, 2008b). In their device, the spatial distribution of the required PCR temperatures can be significantly decreased, resulting in a much smaller chip footprint. In addition, by reducing two-dimensional isothermal areas into one-dimensional isothermal lines, residence time can be eliminated. Very recently, a similar study has been demonstrated by Schaerli et al. (Schaerli et al. 2009). They integrated the circular design of the serpentine channel onto a radial temperature gradient to perform the continuous-flow PCR of single-copy DNA in microfluidic microdroplets. Heating from the center established a natural temperature gradient across the device. The gradient can be externally adjusted via an annular Peltier module that can be heated or cooled according to requirements of the template and primers. Early in 2005, this radial temperature gradient has been used by Cheng et al. for performing the oscillatory-flow PCR where the reagents was pumped back and forth along the radial direction of the chip to achieve rapid temperature cycling, maintaining the flexibility of varying the cycle number and varying the number of temperature zones (Cheng et al. 2005). A similar oscillatory-flow PCR on a linear temperature gradient was recently reported by Ohashi et al. (Ohashi et al. 2007), who used the magnetic transportation to move the PCR reaction droplets to the designated temperature zones on a linear temperature gradient from 50°C to 94°C. Other dynamic flow-based PCR systems induced by a temperature gradient have been reported elsewhere (Braun et al. 2003; Duhr and Braun 2006; Grover et al. 2008; Krishnan et al. 2002; Stoffel et al. 2007). However, all these flow-based PCR systems performed on a temperature gradient do not fall into the category of MG-PCR unless they meet the requirements of the MG-PCR concept described below.
3 Concept of microfluidic gradient PCR (MG-PCR)
4 Experimental section
4.1 Reagents and materials
10 × Taq DNA polymerase buffer (500 mM KCl, 100 mM Tris-HCl (pH 8.8), 0.8% Nonider P-40), MgCl2 solution (25 mM), and thermostable Taq polymerase (5 unit/μl) were purchased from Bio Basic Inc. (BBI) (Ontario, Canada). Deoxynucleotide triphosphate (dNTPs) (10 mM each of dATP, dGTP, dCTP, and dTTP in water), PCR primers, and agarose were obtained from Shanghai Sangon Biological Engineering & Technology Services Co. Ltd. (SSBE) (Shanghai, China). Forward and reverse primer sequences were 5′ -GGA ATC AGG CGT CTG GGT CA-3′ and 5′ -GCC GTT AGT CGC TTC GTC ATA-3′, respectively. The doubly deionized H2O (ddH2O) were purchased from Tiangen Biotech Co. Ltd. (Beijing, China).
Bovine serum albumin (BSA) (fraction V, purity ≥98%, biotechnology grade, Cat. No. 735094), which was used to dynamically passivate the inner surface to decrease the surface adsorption, was purchased from Roche Diagnostics GmbH (Mannheim, Germany). Sodium hypochlorite solution, which was utilized to remove the residual substance from the microchannel before each run, was obtained from Guanghua Chemical Factory (Guangzhou, China). The DNA markers, which contain 2000, 1000, 750, 500, 250, and 100-bp DNA fragments, were bought from Win Honor Bioscience (Beijing, China). GoldViewTM dye was purchased from SBS Genetech Co. Ltd. (Beijing, China). The polytetrafluoroethylene (PTFE) capillary tube (inner diameter 500 μm) was purchased from Wuxi Xiangjian Tetrafluoroethylene Product Co. Ltd. (Wuxi, China). The silicone tube was from Jinan Chen Sheng Medical Silicone Rubber Products Co., Ltd. (Jinan, China).
4.2 Design of temperature gradient platform
4.3 Temperature gradient device assembly and operation
The convective loop reactor was constructed by using the PTFE tubing. After a PTFE tube was fitted into the grooves at the hot and cold part of the temperature gradient device, it was bent to nearly the shape of a “0” (Fig. 3). The length of each vertical side is 40 mm. The total length and volume of the loop are 180 mm and 35 μl, respectively. It is demonstrated later that these dimensions can be further reduced. Lengths of tubing were filled with PCR solutions and then the ends of the tubing were joined by using a sleeve of silicone tube. Slightly overfilling the fluid loop prior to jointing the two open ends can prevent bubble formation. In our prototype, the upper and lower parts of the tube were insulated in order to ensure the two-temperature convective PCR. During each experiment, the arrangement of the heater or the form of temperature gradient facilitated clockwise circulation in the fluid loop, and an ambient temperature of 22°C was maintained, and its variation was within ± 0.5°C. Additionally, the device was inclined to an angle (α) of 60o with respect to the horizontal. Of course, this angle can be varied according to the actual PCR experiments. For example, when a longer DNA template is amplified, the α should be smaller. On the contrary, the amplified DNA segment is shorter, the α should be larger. In addition, when the diameter of the loop is smaller, the α can be adjusted to 90o for use.
After introduction of the solution, the temperature at the bore location was heated to 97°C and maintained 5 min to ensure initial denaturation. Subsequently, a 4–12 V voltage was applied to the fan to execute the convective PCR. After some cycles, the temperature was decreased to 76°C, and then the fan stopped working. This temperature maintained for 6 min to complete final extension.
4.4 PCR experiments
To demonstrate the capability of the MG-PCR, small targets were amplified from E. coli K12 genomic DNA, and the amplified product is a 112-bp fragment out of 1194-bp tyrB gene. Both the control PCR and MG-PCR solutions used for amplification consisted of 0.5 μM of each primer, 0.2 mM of each dNTP, 0.1 unit/μl Taq DNA polymerase, 4 ng/μl DNA templat, 1.5 mM MgCl2, 1 × Taq DNA polymerase buffer and 250 ng/μl BSA. To compare amplification characteristics (speed, specificity, and yield), portions of each PCR solution were amplified in both the MG-PCR device and commercial Mastercycler gradient PCR thermocycler (Eppendorf AG, Hamburg, Germany). To validate the amplification, negative controls (without template DNA or Taq enzyme) were amplified to ascertain if the amplification results from residual contamination. For MG-PCR reactions, the amplification times were 120, 90, 60, 45, and 30 min (not including the initial denaturation and final extension times). In the bench-top PCR thermocycler, two kinds of PCR protocols were used: three-temperature PCR and two-temperature PCR. The former consisted of 3-min denaturation at 94°C, 35 cycles (0.5 min at 94°C, 0.5 min at 60°C, and 1.5 min at 72°C), and final extension at 72°C for 3 min, which gave a total run time of 100 min including temperature ramping times. Two-temperature PCR was programmed for an initial denaturation step of 3 min at 94°C, followed by 35 cycles of 0.5 min at 94°C, 1 min at 60–70°C, and a final extension at 60–70°C for 3 min, with a total run time of 50 min including temperature ramp times. PCR results of positive and negative controls were analyzed on a 1.2 % agarose gel containing GoldViewTM dye. Gels were typically run at 125 V for 35 min.
5 Results and discussion
5.1 Evaluation of temperature gradient
In most of the above-mentioned temperature gradient devices for microfluidic DNA analysis applications, the principle of their working exploits the fact that heat flow in two dimensions between a heat source and cold sink leads to a linear temperature gradient between the two when they are placed in parallel (Cheng et al. 2005; Crews et al. 2007, 2008a, b, c, 2009; Kinahan et al. 2009; Mao et al. 2002a, b; Ohashi et al. 2007; Ross and Locascio 2002; Schaerli et al. 2009). For example, within Ross and Locascio’s work, the temperature gradient was produced by thermally anchoring the thin polycarbonate microchannel chips to alternatively heated or cold blocks (Ross and Locascio 2002). The heated copper block, whose temperature was regulated by a PID controller, was heated using a small high-power resistor embedded into the copper. To regulate the temperature of the cold zones, cold water from a thermostated bath was passed through the holes drilled within the cooled copper blocks. Similar works are also been reported by Mao and coworkers (Kinahan et al. 2009; Mao et al. 2002a, b). Although such systems can easily obtain the desired temperature gradient range, they often require an external bulky water bath and its accessorial equipment (such as temperature controller and circulating pump). As a result, they are difficult to miniaturize onto a single microfuidic device. In order to improve this, some researchers have used the metallic strips and/or thin-film heaters to generate the desired linear temperate gradient (Cheng et al. 2005; Crews et al. 2007, 2008a, b, c, 2009). A network of cooling fins was coupled to the strip corresponding to the cooler zone, and as a result a nearly linear temperature gradient could be obtained (Crews et al. 2008b). However, these experiences maybe showed a general lack of robustness in regulating the temperature gradient. In addition, these systems are complicated in the temperature control system and difficult in establishing stable and uniform temperature gradients with multiple heating sources. Recently, Zhang et al. have developed a novel temperature gradient technology using a slantwise radiative heating system (Zhang et al. 2007c). Although such approach can generate a highly reproducible temperature gradient with high spatial resolution, it lacks the flexibility to changing the temperature gradient.
The average temperature gradient obtained in our device is approximately 1.1°C/mm. This value is higher than that reported by Ohashi and coworkers (0.58°C/mm) (Ohashi et al. 2007). However, the obtained average temperature gradient in the current device is much lower than one reported by Crews et al. (Crews et al. 2008b), that was high up to 3.5°C/mm. We attribute such results to the two following reasons: (1) the thickness of the copper flake and the base of the aluminum fin array is larger. Seen from the aforementioned governing equation, under similar conditions, the temperature difference along the temperature gradient direction on a thin fin flake is apparently larger than that obtained on a thick fin flake. (2) the thermal conductivity of temperature gradient copper plate is higher. Using a material with low thermal conductivity as a heat-conducting plate, such as ceramic, glass, or Teflon, the average temperature gradient should increase. Therefore, it is likely that the temperature difference between the hot and the cold part in our device can be significantly increased or a steep temperature gradient on a compact device can be formed.
5.2 Single-loop PCR in MG-PCR device
5.3 Multi-loop parallel PCR amplification in MG-PCR device
6 Extensions of method
Within the MG-PCR device reported herein, the natural convection pumping mechanism is used to drive the fluid flow in the reaction loop. Although this pump is simple and inexpensive in design, it has some limits in application. For example, the flow rate within the reaction loop is largely dependent upon the inner diameter of the loop and the temperature difference between the PCR temperature zones. Fortunately, a number of mechanical (piezoelectric, pneumatic, and thermopneumatic) and non-mechanical (electrokinetic, magnetohydrodynamic, electrochemical, acoustic-wave, surface tension and capillary, and ferrofluidic magnetic) micropumps have been successfully used within PCR chips (Zhang et al. 2007a). In the future, these micropumps are likely applied to the current MG-PCR device. In addition, as stated earlier, the temperature gradient devices have been successfully applied for many microfluidic DNA analysis applications. Therefore, it is likely that our current temperature gradient device is used for these applications.
We constructed a MG-PCR system with a non-linear temperature gradient formed on a combined finned copper flake. We have shown that the newly developed system is capable of performing parallel PCR amplifications with different annealing temperatures ranging from 60 to 68°C. Compared with the conventional gradient PCR machine, the current MG-PCR device has several obvious advantages, including simple and inexpensive device design, faster amplification rates, flexible incorporation with existing laboratory protocols and fluid handling systems, and easy operation in a portable, low-consumption fashion. Within future MG-PCR device, real-time fluorescence detection or other detection technology such as CE is likely integrated to provide on-line MG-PCR product detection. We expect to be only at the beginning of the MG-PCR development that will have great impact on the field of microfluidic DNA amplification in further years.
This research is supported by the National Natural Science Foundation of China (30700155; 30870676; 30800261), the Program for Changjiang Scholars and Innovative Research Team in University (IRT0829) and the National High Technology Research and Development Program of China (863 Program) (2007AA10Z204).