Reasons and solutions for abnormalities in PCR amplification

Enzyme inactivation: new enzymes need to be replaced, or both old and new enzymes should be used simultaneously to analyze whether false negatives are caused by loss or insufficient enzyme activity. It should be noted that sometimes Taq enzyme or ethidium bromide is forgotten.

Primers: Primer quality, primer concentration, and the concentration of the two primers are symmetrical, which is a common cause of PCR failure or unsatisfactory expansion of the band. Some batches have problems with the quality of primer synthesis. Two primers have a high concentration and a low concentration, resulting in low-efficiency asymmetric amplification. The countermeasures are: 1 Select a good primer synthesis unit. 2 The concentration of the primer should not only look at the OD value, but also pay attention to the primer solution for agarose gel electrophoresis. There must be a primer band, and the brightness of the two primer bands should be roughly the same, such as a primer with a band and a primer without Strips, PCR may fail at this time, and should be resolved in consultation with the primer synthesis unit. If one primer has high brightness and one has low brightness, balance the concentration when diluting the primer. 3 Primers should be stored in high concentration and small amount to prevent multiple freeze-thaw cycles or long-term storage of refrigerators, resulting in failure of primer degradation. 4 Primer design is not reasonable, such as the length of the primer is not enough, the formation of dimers between the primers.

Mg ²⁺ concentration: Mg ²⁺ ion concentration has a great influence on PCR amplification efficiency. Too high concentration can reduce the specificity of PCR amplification. If the concentration is too low, it will affect the PCR amplification yield and even the PCR amplification will not expand. Increase the strip.

Change in reaction volume: The volume used for PCR amplification is usually 20ul, 30ul, 50ul or 100ul. The large volume of PCR is used for the purpose of scientific research and clinical testing. After making a small volume such as 20ul, When you are making a large volume, you must make mold conditions, otherwise it will be easy to fail.

Physical reasons: Denaturation is very important for PCR amplification, such as low denaturation temperature, short denaturation time, and highly likely false negative; annealing temperature is too low, which can cause non-specific amplification and reduce specific amplification efficiency, annealing temperature Excessively high binding primers bind to the template to reduce PCR amplification efficiency. It is sometimes necessary to use a standard thermometer to detect denaturation, annealing and extension temperatures in the instrument or water bath, which is one of the reasons for PCR failure.

Target sequence variation: If the target sequence is mutated or deleted, it affects the specific binding of the primer to the template, or the primer and the template lose complementary sequence due to a certain deletion of the target sequence, and the PCR amplification is not successful.

False positive
The PCR amplification bands appearing are consistent with the target target sequence bands, and sometimes the bands are more tidy and the brightness is higher.

Primer design is not suitable: the selected amplified sequence has homology with the non-target amplified sequence, so when PCR amplification is performed, the amplified PCR product is a non-target sequence. The target sequence is too short or the primer is too short to be prone to false positives. Primers need to be redesigned.

Cross-contamination of target sequences or amplification products: There are two reasons for this contamination: one is cross-contamination of the entire genome or large fragments, leading to false positives. This false positive can be resolved by the following methods: Care should be taken to prevent the target sequence from being drawn into the sample gun or spilled out of the centrifuge tube. All reagents or equipment should be autoclaved except for enzymes and substances that cannot withstand high temperatures. The centrifuge tube and sample tip used should be used at one time. If necessary, the reaction tubes and reagents are irradiated with ultraviolet light to destroy the nucleic acid present before the specimen is added. The second is the contamination of small fragments of nucleic acids in the air. These small fragments are shorter than the target sequences, but have some homology. The splicing can be spliced ​​to each other, and after complementing the primers, the PCR product can be amplified, resulting in the generation of false positives, which can be alleviated or eliminated by nested PCR.

Non-specific amplification band
The bands appearing after PCR amplification are inconsistent with the expected size, either large or small, or both specific amplification bands and non-specific amplification bands. The emergence of non-specific bands is due to the fact that the primers are not completely complementary to the target sequence or the primers polymerize to form a dimer. Second, the Mg ²⁺ 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 the enzyme. Sometimes the enzymes of some sources are prone to non-specific bands and the enzymes of another source do not. Excessive amounts of enzymes sometimes lead to non-specific amplification. The countermeasures are: redesigning the primers if necessary. Reduce the amount of enzyme or exchange enzymes from another source. Reduce the amount of primers, increase the amount of template, and reduce the number of cycles. Appropriately increase the annealing temperature or use two temperature point method (denaturation at 93 ° C, annealing and extension at around 65 ° C).

a sheet-like tow or smear
PCR stripping sometimes occurs with a smear strip or a strip or carpet strip. The reason is often due to the excessive amount of enzyme or the poor quality of the enzyme, the dNTP concentration is too high, the Mg ²⁺ concentration is too high, the annealing temperature is too low, and the number of cycles is too high. The countermeasures are: reducing the amount of enzyme, or transposing an enzyme from another source. 2 reduce the concentration of dNTP. Properly reduce the concentration of Mg ²⁺ , increase the amount of template, and reduce the number of cycles.