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The outstanding feature of the PCR reaction is its large amplification capacity and extremely high sensitivity. In order to improve PCR performance and detection efficiency, we are committed to improving the PCR amplification capacity and detection sensitivity, but the headache is in the process of the experiment. False positives often occur, and a very small amount of sample cross-contamination or PCR product contamination may cause false positives in the experiment.

Five types of PCR product contamination

There are many reasons for PCR contamination, which can be roughly divided into the following categories:

1. Cross-contamination between specimens

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Specimen contamination is mainly caused by the contamination of the container for collecting the specimen, or when the specimen is placed, it is leaked out of the container due to loose sealing, or the specimen is adhered to the outside of the container, which causes cross-contamination; Contamination leads to contamination between specimens; some microbial specimens, especially viruses, can spread with aerosols or form aerosols, resulting in mutual contamination.

2. PCR reagent contamination

The main reason is that during the preparation of PCR reagents, the sample gun, container, double distilled water and other solutions are contaminated by the PCR nucleic acid template.

1.2

3. Cloning plasmid contamination

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In molecular biology laboratories and some laboratories that use cloned plasmids as positive controls, the problem of cloned plasmid contamination is also common. Because the content of cloning plasmid in unit volume is quite high, and more tools and reagents are needed in the purification process, and the plasmid in living cells is very likely to be contaminated due to the strong growth and reproduction ability of living cells.

4. Contamination of amplified products

The contamination of amplified products is the most common contamination problem in PCR reactions. Because the copy quantity of PCR product is large (generally 1013 copies/ml), which is far higher than the limit of PCR detection copy number, a very small amount of PCR product contamination can cause false positives.

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5. Aerosol pollution

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Aerosol pollution is the most likely form of contamination of PCR products, and it is also the easiest to be overlooked. It is formed by the friction between the liquid surface and the air. Generally, aerosol contamination can be formed when the lid is opened, when the sample is aspirated, or even when the reaction tube is shaken vigorously. According to calculations, an aerosol particle can contain 48,000 copies, so the pollution caused by it is a problem that deserves special attention.

In particular, testing laboratories often use the same pair of primers to test a certain gene. Over time, a large amount of PCR product contamination will occur in the laboratory space. Once such contamination occurs, it is difficult to eliminate it in a short time.

For the first three types of pollution, we can use effective means to avoid, but the pollution caused by PCR products is difficult to prevent, especially in the construction of non-standard PCR laboratories. In the PCR process, when the pipette tip sucks and blows the liquid, and the PCR tube cover is opened, an aerosol will be formed. The DNA molecules carried by the aerosol (one aerosol can carry tens of thousands of DNA) are difficult to eliminate because they float in the air. Once the next round of PCR experiments are introduced, false positives will inevitably occur.

As shown in the figure below, the negative control also amplified the corresponding band of interest:

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The first part of this issue of PCR pollution and prevention is introduced here. The next issue will bring you the second part "Prevention of PCR product contamination", so stay tuned!


Post time: Jul-25-2017