Causes of abnormal PCR amplification bands and solutions

When using PCR, the most frequently encountered problem is that there will be differences in amplification. Take this opportunity to discuss with you the reasons and solutions for the out-of-time PCR amplification strips.

False negative, no amplified band

The key steps of PCR reaction are ① preparation of template nucleic acid; ② primer quality and specificity; ③ enzyme quality; ④ PCR cycle conditions. Finding the reasons should also be analyzed and researched for the above links.

Template: ①The template contains miscellaneous protein; ②The template contains Taq enzyme inhibitor; ③The protein in the template is not digested, especially the histone in the chromosome; ④The template is lost too much or phenol is inhaled when the template is extracted and prepared; ⑤ The template nucleic acid is not completely denatured. When the quality of the enzymes and primers is good, no amplified bands will appear. It is most likely that the sample is digested, and the template nucleic acid extraction process is faulty. Therefore, it is necessary to prepare an effective and stable digestion solution. The program should also be fixed and should not be changed at will .

Enzyme inactivation: It is necessary to replace with a new enzyme, or use both the old and the new enzyme at the same time to analyze whether the enzyme activity is lost or not enough to cause false negatives. It should be noted that sometimes forgot to add Taq enzyme or ethidium bromide.

Primers: The quality of primers, the concentration of primers, and whether the concentrations of the two primers are symmetrical are common reasons for PCR failure or unsatisfactory amplified bands and easy diffusion. The quality of the primer synthesis of some batches is problematic. 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: ①Select a good primer synthesis unit. ②The concentration of the primer depends not only on the OD value, but also pay attention to the primer stock solution for agarose gel electrophoresis. The primer band must appear, and the brightness of the two primer bands should be roughly the same. Strips, PCR may fail at this time, and it should be resolved through consultation with the primer synthesis unit. If one primer has high brightness and one has low brightness, balance its concentration when diluting the primer. ③Primers should be stored in small amounts at high concentration to prevent multiple freeze-thaw cycles or long-term storage in the refrigerator, which may cause deterioration and degradation of the primers. ④ The primer design is unreasonable, such as the length of the primer is not enough, the formation of dimer between the primers, etc.

Mg2+ concentration: Mg2+ ion concentration has a great influence on PCR amplification efficiency. Too high concentration can reduce the specificity of PCR amplification, while too low concentration will affect PCR amplification yield and even make PCR amplification fail without showing amplified bands.

Change of reaction volume: Usually the volume used for PCR amplification is 20ul, 30ul, 50ul or 100ul. The volume used for PCR amplification is set according to the different purposes of scientific research and clinical testing. After making a small volume such as 20ul, When making a large volume, the conditions must be modelled, otherwise it is easy to fail.

Physical reason: Denaturation is very important for PCR amplification. For example, if the denaturation temperature is low and the denaturation time is short, false negatives are very likely; if the annealing temperature is too low, it can cause non-specific amplification and reduce specific amplification efficiency. Annealing temperature Too high affects the combination of primer and template and reduces PCR amplification efficiency. Sometimes it is necessary to use a standard thermometer to check the denaturation, annealing and extension temperatures in the thermal cycler or the water bath. 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 and the template, or the primer and template lose their complementary sequence due to the deletion of a certain segment of the target sequence, the PCR amplification will not be successful.

False positive

The PCR amplified bands appearing are consistent with the target target sequence bands, and sometimes the bands are more neat and brighter.

 Inappropriate primer design: The selected amplified sequence has homology with the non-target amplified sequence. Therefore, the amplified PCR product is a non-targeted sequence during PCR amplification. If the target sequence is too short or the primer is too short, false positives are prone to occur. Need to redesign the primers.

 Cross-contamination of the target sequence or amplification product: There are two reasons for this type of contamination: one is the cross-contamination of the entire genome or large fragments, leading to false positives. This false positive can be solved by the following method: the operation should be careful and gentle to prevent the target sequence from being sucked into the sample gun or spilled out of the centrifuge tube. Except for enzymes and substances that cannot withstand high temperatures, all reagents or equipment should be autoclaved. All centrifuge tubes and sample injection tips should be used once. When necessary, the reaction tube and reagents are irradiated with ultraviolet rays before adding the specimens to destroy the existing nucleic acids. The second is the contamination of small fragments of nucleic acid in the air. These small fragments are shorter than the target sequence but have a certain degree of homology. It can be spliced to each other, and after complementing the primers, PCR products can be amplified, which can lead to false positives. Nested PCR can be used to reduce or eliminate them.

Non-specific amplification band

 The bands appearing after PCR amplification are inconsistent with the expected size, large or small, or both specific amplified bands and non-specific amplified bands appear at the same time. The reasons for the appearance of non-specific bands are as follows: First, the primer and the target sequence are not completely complementary, or the primer polymerizes to form a dimer. The second 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 is the quality and quantity of enzymes. Often, enzymes from some sources are prone to non-specific bands while enzymes from another source do not. Too many enzymes sometimes cause non-specific amplification. The countermeasures are: redesign the primers when necessary. Reduce the amount of enzyme or switch to another source of enzyme. Reduce the amount of primers, increase the amount of template appropriately, and reduce the number of cycles. Properly increase the annealing temperature or use the two-temperature point method (93°C denaturation, 65°C annealing and extension).

 Flaky drag or smear tape appears

In PCR amplification, smear bands, flaky bands, or carpet-like bands sometimes appear. The reason is often due to the excessive amount of enzyme or poor quality of the enzyme, too high dNTP concentration, too high Mg2+ concentration, too low annealing temperature, and too many cycles. The countermeasures are: reduce the amount of enzymes, or exchange enzymes from another source. ②Reduce the concentration of dNTP. Appropriately reduce the Mg2+ concentration, increase the amount of template, and reduce the number of cycles.