PCR FAQ 1. False negative, no amplification band appears 2. False positive 3. Non-specific amplification bands appear 4. Flaky drag strips or smear strips appear：
1False negative, no amplified band appears The key aspects of the PCR reaction are
① preparation of template nucleic acid
② primer quality and specificity
③ enzyme quality and
④ PCR cycle conditions. To find the reasons, analysis and research should also be conducted on the above links.
Template:
① The template contains impurity proteins
② The template contains Taq enzyme inhibitors
③ The proteins in the template are not digested and removed, especially the histones in chromosomes
④ Too much was lost during the extraction and preparation of the template, or phenol was inhaled
⑤The template nucleic acid is not completely denatured. When the quality of enzymes and primers is good, if amplification bands do not appear, it is most likely that there is something wrong with the digestion process of the specimen or the template nucleic acid extraction process. Therefore, an effective and stable digestion solution must be prepared, and the procedure should be fixed and should not be changed at will. . Enzyme inactivation: It is necessary to replace the new enzyme, or use both old and new enzymes at the same time to analyze whether false negatives are caused by loss or insufficient enzyme activity. It should be noted that sometimes the Taq enzyme is forgotten.Primers: Primer quality, primer concentration, and whether the concentrations of the two primers are symmetrical are common reasons for PCR failure or unsatisfactory amplification bands and easy diffusion. There are problems with the quality of primer synthesis in some batches. One of the two primers has a high concentration and the other has a low concentration, resulting in low-efficiency asymmetric amplification.
The countermeasures are:
① Select a good primer synthesis unit.
② The concentration of primers should not only look at the OD value, but also pay attention to the primer stock solution for agarose gel electrophoresis. There must be primer bands, and the brightness of the two primer bands should be roughly the same. For example, one primer has a band and the other primer has no band. For strips, PCR may fail at this time and should be resolved through negotiation with the primer synthesis unit. If one primer has high brightness and the other has low brightness, balance the concentrations when diluting the primers.
③ Primers should be stored in high concentration and small quantities to prevent repeated freezing and thawing or long-term storage in the refrigerator, which may lead to deterioration and degradation of the primers.
④The primer design is unreasonable, such as the primer length is not enough, dimers are formed between primers, etc. Mg2+ concentration: Mg2+ ion concentration has a great influence on PCR amplification efficiency. If the concentration is too high, it can reduce the specificity of PCR amplification. If the concentration is too low, it will affect the PCR amplification yield and even cause the PCR amplification to fail without amplifying bands. Changes in reaction volume: Usually the volumes used for PCR amplification are 20ul, 30ul, and 50ul. Or 100ul, what volume should be used for PCR amplification is set according to different purposes of scientific research and clinical testing. After making a small volume, such as 20ul, and then making a larger volume, you must follow the conditions, otherwise it will easily fail. Physical reasons: Denaturation is very important for PCR amplification. If the denaturation temperature is low and the denaturation time is short, false negatives are very likely to occur; the annealing temperature is too low, which can cause non-specific amplification and reduce the specific amplification efficiency. The annealing temperature is too high. Highly affects the binding of primers to templates and reduces PCR amplification efficiency. Sometimes it is necessary to use a standard thermometer to check the denaturation, annealing and extension temperatures in the amplifier or water-soluble pot. This is also one of the reasons for PCR failure. Target sequence variation: If the target sequence is mutated or deleted, which affects the specific binding of the primer to the template, or the primer and template lose the complementary sequence due to the deletion of a certain segment of the target sequence, the PCR amplification will not be successful.
2.false positive The PCR amplification band that appears is consistent with the target sequence band, and sometimes the band is more orderly and brighter. Inappropriate primer design: The selected amplification sequence has homology with the non-target amplification sequence, so when performing PCR amplification, the amplified PCR product is a non-target sequence. If the target sequence is too short or the primer is too short, false positives may easily occur. Primers need to be redesigned. Cross-contamination of target sequences or amplification products: There are two reasons for this contamination: First, cross-contamination of the entire genome or large fragments, leading to false positives. This false positive can be solved by the following methods: Be careful and gentle when operating to prevent the target sequence from being sucked into the sample gun or splashed out of the centrifuge tube. Except for enzymes and substances that cannot withstand high temperatures, all reagents or equipment should be sterilized by high pressure. All centrifuge tubes and sample injection pipette tips should be used once. If necessary, the reaction tubes and reagents are irradiated with ultraviolet light before adding the specimen to destroy the nucleic acids present. The second is the contamination of small fragments of nucleic acids in the air. These small fragments are shorter than the target sequence, but have certain homology. They can be spliced to each other, and after being complementary to the primers, the PCR products can be amplified, resulting in false positives, which can be reduced or eliminated by nested PCR methods.
3.Non-specific amplification bands appear The bands that appear after PCR amplification are inconsistent with the expected size, either larger or smaller, or both specific amplification bands and non-specific amplification bands appear at the same time. The reasons for the appearance of non-specific bands are: first, the primers are not completely complementary to the target sequence, or the primers aggregate to form dimers. The second reason is that the Mg2+ ion concentration is too high, the annealing temperature is too low, and the number of PCR cycles is too high. The second factor is the quality and quantity of the enzyme. Enzymes from some sources are often prone to non-specific bands but enzymes from other sources do not. Excessive amounts of enzymes can sometimes lead to non-specific amplification. The countermeasures include: redesign primers if necessary. Reduce the amount of enzyme or replace it with another source. Reduce the amount of primers, increase the amount of template appropriately, and reduce the number of cycles. Increase the annealing temperature appropriately or use the two-temperature point method (denaturation at 93°C, annealing and extension around 65°C).
4.Flaky drag or smears appear PCR amplification sometimes appears as smeared bands, sheet-like bands or carpet-like bands. The reasons are often caused by too much enzyme or poor quality of enzyme, too high dNTP concentration, too high Mg2+ concentration, too low annealing temperature, and too many cycles. The countermeasures include: ① Reduce the amount of enzyme, or replace the enzyme with another source. ②Reduce the concentration of dNTP. Appropriately reduce the Mg2+ concentration. Increase the amount of templates and reduce the number of cycles