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A Comprehensive Explanation of PCR Technology Principles and Fluorescent PCR Methods

A Comprehensive Explanation of PCR Technology Principles and Fluorescent PCR Methods

Ⅰ. The principle of PCR technology

This year, the COVID-19 is raging around the world, and everyone must have heard of nucleic acid testing. In the nucleic acid detection of the epidemic, PCR fluorescence technology plays a central role, which can help inspectors quickly and accurately determine whether they are infected with the COVID-19.

So what exactly is PCR technology?

PCR is an acronym for Polymerase Chain Reaction. In short, PCR consists of three basic reaction steps: high temperature denaturation - low temperature annealing - medium temperature extension:

1. Denaturation of DNA to become single-stranded

After the template DNA is heated to about 95°C for a certain period of time, the double-strand of the template DNA is dissociated into a single-strand so that it can bind to the primer and prepare for the next round of reaction.

2. Low temperature annealing

After the template DNA is denatured into single strands, the temperature is lowered to about 55°C, and the primers are paired with the complementary sequences of the template DNA single strands.

3. Medium temperature extension

Under the action of Taq enzyme, the DNA template and primer conjugates use dNTP as the reaction raw material and the target sequence as the template, according to the principle of base pairing and semi-reserved replication, and finally synthesize a new semi-reserved replication chain complementary to the template DNA chain. .

Ⅱ. Detection methods of fluorescent PCR

At present, most of the COVID-19 detection reagents approved by the State Drug Administration of China use fluorescent PCR methods to detect the COVID-19.

The fluorescent PCR method refers to adding fluorescent groups to the PCR reaction system. With the progress of PCR amplification, the reaction products continue to accumulate, and the fluorescence signal intensity also increases proportionally. Fluorescence amplification curve graph, real-time detection of PCR fluorescence process.

Ⅲ. Principles of selecting PCR fluorescent consumables

1. Material

PCR is generally made of PP material, because PCR/qPCR consumables are usually in direct contact with reagents or samples, while polypropylene (PP) material is a biologically inert material, the surface is not easy to adhere to biomolecules, and has good chemical resistance and temperature. Tolerance (can be autoclaved at 121°C and can also withstand temperature changes during thermal cycling). PCR consumables are all PP material.

2. PCR tube/plate volume

How to choose PCR tubes/plates of different volumes?

Selection purpose: select the appropriate product according to the specific experimental requirements. The volume of PCR tubes can mostly meet the requirements of PCR reactions. PCR tubes are ideal for low to medium throughput PCR/qPCR experiments, with single tubes and strips being the two most commonly used formats.

A single tube provides the flexibility to set the exact number of reactions to run. Alternatively, a single 0.5mL tube can be selected for larger reaction volumes.

Couplings are usually available in 8 or 12-tube format with separate or with caps. Compatible with single- and multi-channel pipettes for low- and medium-throughput experiments. Strips with caps open and close sample tubes independently to prevent sample contamination.

3. Wall thickness

Tube wall thickness directly affects thermal conductivity, and extremely thin wall thicknesses optimize heat transfer and reduce cycle time. The ultra-thin-wall plastic is about 50% thinner than the standard version, further reducing the thermal barrier, resulting in a faster and more robust response. PCR consumables are all ultra-thin tube walls.

4. Color

PCR plates are often available in a variety of different color formats to facilitate visual differentiation and identification of samples, especially in high-throughput experiments. For ordinary PCR reactions, it is possible to use transparent or colored PCR tubes, and transparent PCR tubes are more helpful for sample classification management.

Although the color of the plastic has no effect on DNA amplification, high-quality white PCR consumables are recommended for qPCR. Since qPCR requires real-time quantitative detection of fluorescence signal intensity, sensitive and accurate fluorescence signal transmission is required. Experiments have shown that, compared with traditional transparent PCR products, white PCR products can reflect the fluorescent signal to the greatest extent, reduce the cross-contamination of space signals, and obtain the results of real-time fluorescence quantitative PCR.

Different brands of instruments, due to the different position design of the fluorescence detector, please refer to the materials recommended by the manufacturer. For the Bio-Rad CFX or Roche 480 real-time PCR instrument, the manufacturer recommends using white PCR plates. Because the PCR plate is either white or transparent, and has various specifications, it is suitable for common PCR instruments on the market.