Dry goods | Inhibitors in a sample that affect PCR detection
To successfully convert a sample into a PCR result, there are many steps that need to be paid attention to in the early stage, including: sample collection, transportation, storage, and nucleic acid extraction. Each step needs to be careful, and the middle process can be described as difficult. In the process of converting samples into PCR results, what factors make the conversion process so difficult?
1. The source of PCR inhibitors
Sources of PCR inhibitors include endogenous and exogenous
1. Endogenous
Naturally occurring constituents in specimens. Such as: immunoglobulin, protease, hemoglobin and its metabolites, hemoglobin lactoferrin, myoglobin, lipids, mucin, urea, ions, bile salts, polysaccharides, etc.
2. Exogenous
Externally introduced inhibitors. Such as: heparin anticoagulant, cellulose and nitrocellulose, glove talc, inhibitors contained in specimen containers or sampling equipment
2. General mechanism of PCR inhibitors
The mechanism of most of the PCR inhibitors is not fully understood, but can basically be classified into three categories:
✦1. Interference with cell lysis during nucleic acid extraction;
✦2. Degrade or wrap nucleic acid;
✦3. The thermostable DNA polymerase is inactivated.
1. Interference with cell lysis during nucleic acid extraction
Cells are lysed, and nucleic acid is released so that nucleic acid interacts with enzyme to produce amplification. If cell lysis is not complete and nucleic acid release is not complete, there will be no amplification. Common simple treatment: boiling, advantages: time-saving, simple operation. Disadvantages: sometimes the DNA released by boiling cannot be completely separated from the structural protein or DNA binding protein, resulting in amplification inhibition. The method of boiling alone will reduce the amplification sensitivity.
2. Inactivation of thermostable DNA polymerase
Humic compounds are the most common inhibitors in environmental samples, which may inhibit polymerase activity through chelation of Mg2+.
3. Degrade or encapsulate nucleic acid
DNA can be degraded due to some physical, chemical, or enzymatic factors. For example, when stored amplified products are electrophoresed after amplification, the bands sometimes appear blurred because of the degradation of DNA. The primary structure of DNA is unstable and will be degraded due to hydrolysis, non-enzymatic methylation, oxidative damage, and enzymatic degradation.
Nuclease is present in the cell. If the nuclease treatment is not clean, it will remain in the purified nucleic acid sample, thereby degrading the target nucleic acid. Restriction enzymes produced by microorganisms such as bacteria can also be a factor that degrades DNA. Some bacterial DNA enzymes are thermostable nucleases, which can hydrolyze genome and primer DNA in the mid-stage of amplification.
Nucleic acids and primers also fail to amplify because they cannot be combined with DNA polymerase. For example, the inhibitory effects of proteins, polysaccharides, cell debris, lipids, etc. on PCR are likely to be caused by physical packaging of DNA to prevent it from contacting polymerase.
Talc powder on latex gloves may affect PCR amplification through its non-specific binding to DNA. Because DNA can be bound to glass, silica, etc., this is also the basic principle of using silicon adsorption methods to extract nucleic acids.
After understanding nucleic acid inhibitors, how can we reduce the impact of these inhibitors on the results? The main technique is nucleic acid extraction. Nucleic acid is divided into DNA and RNA, and extracting it from the sample is the prerequisite for PCR. The purpose of extracting and purifying nucleic acids from complex samples is:
✦1. Remove PCR inhibitors;
✦2. Increase the concentration of target nucleic acid so that it can reach the measurement range of a specific PCR;
✦3. Increase the uniformity of the sample to ensure the precision and repeatability of the measurement.
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