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Polymerase Chain Reaction (PCR)

Polymerase chain reaction (PCR) technology is the process of chemically amplifying low concentration DNA samples. This method can amplify several copies of template sequences or even one DNA molecule within a few hours7~108It is easy to operate and has been widely used in various fields of molecular biology research, with the core being semi preserved replication of DNA.

PCR technology is mainly divided into three stages:

（1）DNA denaturationHeat the double stranded DNA sample at 92-97 ℃ to break the hydrogen bonds between the strands and decompose them into two single stranded molecules.

（2）annealingQuickly lower the temperature to 55-65 ℃, at which point the single stranded DNA and primer complement each other according to the principle of base pairing.

（3）extendRaise the temperature to around 72 ℃ for DNA extension reaction.

After each cycle of denaturation, annealing, and extension, the DNA content doubles. In PCR reactions, temperature changes play a decisive role in DNA replication efficiency: the faster the heating and cooling process, the shorter the reaction time required.

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Molecular POCTproduct

I'm sure everyone still remembersVisby Medical Test，This home self checking card box,numbercall, name, say, weighPCR testing without the need for equipmentAgent（Actually, the equipment was put into a cardboard box）It has many advantages:Compact, portable, easy to operate, but a bit expensive.

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The internal structure of the product can be seen from the picture, and the red arrow indicates the topic we are sharing today.

VisbyThe amplification module of the home self checking card box is“PCR microfluidic chip”More accurately, it should be referred to asContinuous flow PCR microfluidic chipBy using the method of "space and time conversion", the reaction process is achieved by driving the mixed sample through different temperature zones.

“VisbyThe success of home self inspection card boxes is attributed to the use of microfluidic technology. ”

MicrofluidicscontrolThe heat capacity of the chip is small, the specific surface area is large, and the heat transfer efficiency is much higher than that of macroscopic PCR devices. Therefore, it can use the rapid heating and cooling process to achieve rapid amplification of DNA. Its advantages not only save sample consumption, but also improve the efficiency and sensitivity of detection, and achieve portable detection.

All reactions need to be completed in a liquid environment and involve precise temperature control, making microscale flow and heat transfer issues particularly important in PCR systems（It should be pointed out that in addition to the three-step PCR reaction, there are also two-step reactions that combine annealing and extension into one step,VisbHome self inspection card box is a two-step reaction）.

PCR microfluidic chips can be classified according to their different structures微室 PCR（micro-chamber PCR）andContinuous PCR (Continuous PCR)Two basic modes.The difference between the two lies in:

(1) The temperature change of microcavity PCR occurs within a fixed volume, and the temperature change is dynamically controlled by the external environment;

(2) Continuous flow PCR achieves passive temperature switching by flowing liquid through positions with different temperature distributions.

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Microchamber PCRmicrofluidic chip

advantageHaving a small volume, it is easy to achieve multifunctional integration.

shortcomingThe cooling rate is limited by the heat capacity of the chip itself, whileMicrochamber PCR microfluidic chips have a large "dead volume" in structure, making cleaning difficult. They can usually only be used once and are not suitable for continuous amplification reactions with multiple reagents.

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continuousFlow PCR microfluidic chippiece

advantageContinuous flow PCR microfluidic chip with the help ofBy using the method of "space and time conversion", the reaction process is achieved by driving the mixed sample through different temperature zones. Due toContinuous flow PCRmicrofluidic chipThe reaction mainly relies on microchannels, with a relatively small fluid "dead volume". By using appropriate buffer solutions for spacing and flushing, cross contamination between different samples can be avoided, enabling continuous flow PCR microfluidic chips to achieve continuous injection and batch processing of multiple samples, thus having the function of an "on-site chemical amplifier".

shortcomingContinuous flow PCRmicrofluidic chipThe drawback is that the number of amplifications depends on the number of times three temperature zones are passed, and once the channel design scheme is determined, this cycle cannot be changed.

Some emerging technologies can overcome the shortcomings of the above two systems, for example, research has shown that they canslightRoom PCRmicrofluidic chipAdding metal nanoparticles to the liquid medium and heating the metal particles with isomorphic microwaves, thus heating the entire fluid medium, has the characteristics of high heating efficiency and flexible control mode.

There are various temperature control methods for microfluidic PCR devices. Krishnan et al. designed a microscale PCR reactor utilizing the Rayleigh Bernard convection effect, and its structure is shown in the following figure.

Microscale PCR reactor based on Rayleigh Bernard convection effect

The device is heated to 97 ℃ with a hot plate at the bottom of the microreactor and maintained at 61 ℃ with a water-cooled plate at the top. Due to the temperature difference, buoyancy convection is generated inside the microreactor, which can form stable Rayleigh Bernard convection under specific conditions. DNA molecules and other reaction components in the solution circulate between 97 ℃ and 61 ℃ as they flow, thus completing two-step PCR amplification of DNA. This device can change the flow mode by altering the geometric dimensions of the intermediate reaction vessel.

Hu et al. utilized the high conductivity of PCR amplification solution to generate different Joule heats through electric fields of different intensities, achieving temperature control in microchannels and thus completing DNA PCR amplification.

PCRdeviceIn terms of gender transitionretreatFire/Extension StepSuddenlyTemperature distribution

References:

[1]Guoqing Hu, Qing Xiang, Rachel Fu, Bo Xu, Roberto Venditti, Dongqing Li. (2006). Electrokinetically controlled real-time polymerase chain reaction in microchannel using Joule heating effect. Analytica Chimica Acta, 557, 146-151.

[2] Madhavi Krishnan, Victor M. Ugaz, Mark A. Burns. (2002).PCR in a Rayleigh-Benard Convection Cell. Science, 298, 793.