Nucleic Acid Amplification

Nucleic acid-based technology (NAT) plays an important role in molecular diagnosis. However, most research still relies on manual analysis of individual samples by skilled technicians, which is time-consuming and labor-intensive. In recent years, with the advancement of microfluidic design, integrated miniature full analysis systems have emerged to overcome the limitations of traditional detection methods. These high-throughput microfluidic systems can quickly perform experiments in parallel, and can complete NAT analysis within hours or even minutes.

One of the key issues of NAT is nucleic acid amplification. At present, various nucleic acid amplification techniques have been developed, which are usually divided into isothermal and non-isothermal methods. Microfluidic technology can be adjusted for both methods to achieve rapid amplification.

Polymerase Chain Reaction on A Chip

Polymerase chain reaction (PCR) is a non-isothermal method. PCR requires the mixing of several reagents including DNA samples, primers, nucleotides, and DNA polymerase. Once the mixture is prepared, the temperature needs to be changed in each cycle to trigger denaturation, annealing, and extension. Therefore, the reagents in the chip need to undergo several rounds of thermal cycling. Generally, there are two methods for performing PCR reactions on a chip, namely transient PCR and spatial domain PCR. In this process, the reagent is either fixed or flowing.

Spatial PCR

The sample continuously moves through a fixed temperature area in the microchannel, and thermal cycling occurs in space. This category is called a spatial or continuous flow PCR device. The advantages of heat transfer make this concept attractive for designing microfluidic PCR devices. Because only the sample block is periodically heated and cooled, the thermal inertia of the system is reduced. Therefore, the time interval between temperature transitions is related to the sample flow rate and thermal equilibrium time, thereby achieving rapid thermal response and short reaction time.

Two common methods in flow-through nucleic acid amplification are oscillating flow and serpentine channel.

Microfluidic device for serpentine space PCR	The sample is pumped through a single serpentine channel etched in the glass chip, thereby heating or cooling the sample through a predetermined temperature zone to mimic a PCR cycle. At a fixed flow rate, the residence time in a given temperature zone is determined by the channel cross-section and the length of the channel section.
Microfluidic device for oscillating flow PCR	PCR reagents are transported back and forth through a single simple straight channel that spans various temperature zones. A pump is used to circulate chemicals back and forth between temperature zones. Parallel channels can be used to increase throughput. The oscillating motion can be simply controlled to allow a longer residence time in any temperature zone.

Transient PCR

The transient method introduces the PCR solution into a single or multiple reaction chambers. Then, the chamber is subjected to repeated heating and cooling processes corresponding to the thermal cycle curve. Since the sample is fixed and its temperature changes over time, the amplification method is called transient PCR. In this type of equipment, the temperature profile does not depend on the channel design, so the heating and cooling procedures can be modified to modify the PCR thermal cycling conditions. No sample transportation is required, so no pump is required.

Fig.1 (A) Microfluidic design of a compact disc (CD) device and schematic illustration of single-cell isolation. (B) (a) Schematic graph of the multi-layered centrifugal disc. The disc is comprised of five layers of hard plastic. (b) (Right) Image of the integrated centrifugal microfluidic platform for pumping, valving, and thermocycling of fluid. (Left) Close-up of the actuated thermal platform showing the location of the central thermocycling TE and two ice-valve TEs. (c) Schematic presentation of the hardware details and fluidic process for the integrated CD system. (Gorgannezhad L, et al. 2018)

There are many ways to heat and cool the chip. The heater can be placed around the chamber to heat and cool the reagent, or the internal electrode heater can be used to overcome the time-consuming caused by the external heater. Electrodes are placed in the chamber and connected to a power source to heat the chamber. A common method is to print electrodes on a glass slide and bond them to the chip to form an on-chip heating module. The temperature changes by changing the current and voltage.

Microfluidic Device Based on Isothermal Amplification

Although PCR is the most popular method of DNA amplification, the technique requires several thermal cycles. The isothermal amplification of NA eliminates the need for thermal cycling steps, thereby achieving low cost and high detection quality. In the past decade, a variety of isothermal methods for NA amplification have been developed. Isothermal methods integrated with microfluidics include but are not limited to:

• Rolling circle amplification (RCA）
• Strand Displacement Amplification (SDA)
• Cycle-mediated isothermal amplification (LAMP)
• Nucleic acid sequence-based amplification (NASBA)

Does your research focus on nucleic acid amplification? Please contact us for more information about purchasing microfluidics for nucleic acid amplification research.

References

• Gorgannezhad L, et al. (2019). "Microfluidic-Based Nucleic Acid Amplification Systems in Microbiology." Micromachines (Basel). 10(6): 408.
• Chang C. M, et al. (2019). "Nucleic Acid Amplification Using Microfluidic Systems." Lab Chip. 13: 1225-1242.

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