【 5-Minute Explanation of Experiments 】 A Review of Factors Affecting PCR Experiment Results

Article source: Jinglai Biology

PCR only requires a few DNA molecules as templates to achieve large-scale amplification. It is important to prevent the reaction system from being contaminated or cross-contaminated by a small amount of DNA templates, as this is the greatest possibility of false positives.

1. Wash your hands thoroughly before using the PCR room. Wear gloves when operating and change them frequently.

2. Complete sets of reagents should be aliquoted in small quantities and stored separately to prevent reuse. Prepare reagents with new equipment and dispose of them once after use.

3. After use, remember to turn on the ultraviolet for 15 to 30 minutes, especially the liquid preparation chamber. After the experiment is completed, the countertop should be cleared up. This is particularly important for PCR experiments because the experimental waste may be a source of contamination for others. Even if it is not, the undiscarded waste can easily become a carrier of DNA aerosols that may exist in the air, causing contamination.

4. Before using the reagent tube, perform an instantaneous centrifugation (10 seconds) to allow the liquid to settle at the bottom of the tube, reducing the chance of contaminating the gloves and the pipette.

5. When adding the template, be sure to avoid spray contamination. When drawing liquid, try to avoid drawing it too quickly with force. Avoid spraying reaction components or templates onto the pipette to prevent contamination of the pipette and the environment. All non-ready-to-use tubes should be tightly covered. Gloves should be changed after adding template DNA.

6. Accurately absorb each reaction component. When preparing the reaction solution, try to avoid adding 1-2 μL of it and avoid preparing a single 20μL reaction system. High-concentration liquids should be diluted to an appropriate concentration before application and then added to the reaction system. When conducting multi-tube PCR reactions, first mix all the components except the template together, and then divide them into individual PCR reaction tubes. In this way, the amount of each component added is more accurate.

7. Precautions during electrophoresis: When loading samples for electrophoresis, avoid the PCR products drifting to other Wells to prevent them from affecting the result determination. Special attention should be paid to the fact that EB is a strong carcinogen. Gloves must be worn and the operation should be carried out with caution. Do not splash it when shaking.

Both the negative control and the positive control showed positive amplification bands

Reason

① Contamination of negative samples and PCR reaction tests.

When loading samples for electrophoresis, avoid the PCR products drifting to other Wells to prevent them from affecting the result determination.

Solution

① Replace all reagents and, if necessary, all pipettes.

② Careful operation should be carried out, or the agarose gel can be thickened to increase the capacity of each well, which can prevent the overflow of the sample solution.

2. The positive control turns negative

Reason

① Positive samples or the addition of positive templates have failed. Whether it is tissue or serum, the number of pathogens is significantly reduced after one freeze-thaw cycle.

② The dosage of the added test is inaccurate or a certain component is forgotten.

Solution

① Divide the positive control samples into small packages. It is best if each package is sufficient for one use.

② Check carefullyCheck the records and calibrate the pipette.

3. The amplification band of the positive control is weak

Reason

The content of nucleic acid dyes in agarose during electrophoresis is low or it has become ineffective if used after a long time.

The reasons for the low amplification efficiency are:

① Problems with the reaction system: Inaccurate addition of buffers, TaqDNA polymerase, primers, etc. during operation leads to the failure to achieve the optimal reaction system.

② The instrument is not working properly.

③ It is caused by the presence of a considerable amount of PCR reaction inhibitors remaining during the sample processing. In conclusion, in most cases, it is caused by the operator's fear that the PCR test will not yield a positive result, intentionally or unintentionally increasing the sample volume for PCR testing.

Countermeasures

① Place the agarose gel in the EB electrophoresis solution and stain it for another 30 minutes before observing.

② Check whether the instrument is working properly.

③ Increase the dilution factor for purifying DNA or cDNA samples.

4. Non-specific amplification bands appeared (in addition to the positive bands, many amplification bands appeared in the sample)

Reason

① There is too much sample template.

② There are many primers or TaqDNA polymerases.

③ The Mg²+ concentration is too high or the annealing temperature is too low.

Countermeasures: Based on the specific circumstances of the experiments, analyze the possible causes mentioned above and take corresponding measures.

5. The following measures can be taken to avoid false positives and contamination

5.1 Compartment (area) Operations: Sample processing includes sampling, nucleic acid extraction and reagent preparation

PCR reactions and product identification should be carried out in separate rooms (areas) and by different individuals. This is because contamination may occur through frequent atomization in the samples, such as boiling, shaking, and centrifugation. The diameter of atomized particles can reach 20μm, and particles larger than this size can contain 4,000 copies of amplification products. Therefore, it is advisable to operate within a fume hood as much as possible.

The most important measure to prevent contamination is that the PCR preparation process should be completely separated from any operations related to the PCR amplification products to avoid contact between the amplification products and the reaction items.

5.2 Control: Negative and positive controls should be set up for each PCR reaction.

Negative controls should include redistilled water without DNA and known negative control DNA controls. A positive control is a simple way to determine the result. Especially when starting a certain PCR or establishing a method, the amplification products of the positive control may become the root cause of false positives in subsequent experiments.

Therefore, it is currently believed that once the method is mature, positive subjects will no longer be set. The results can be determined by the size of the amplified fragments or the southern hybridization method. For the detection of exogenous DNA such as microbial DNA, relevant DNA clone fragments or genomes can be used as positive controls.

If not, a positive specimen from a book can be used as a substitute.

5.3 Other Operations:

PCR amplification and sample processing are best carried out by different operators. The operators should wear gloves, hats, and masks and isolation gowns as much as possible.

Reagent operations should be minimized as much as possible, and the operation of secondary amplification, namely PCR, should be particularly strict. When used for detection, the number of cycles should be minimized as much as possible.

Centrifuge tubes and tips related to PCR should be used only once as much as possible. When opening centrifuge tubes, adding samples and taking samples, be gentle to avoid contamination of the pipette head. All PCR-related reagents should be packed in small tubes. Try not to use them repeatedly. Once there is evidence of contamination, they should be discarded immediately.

6. The following measures can be taken for false negatives

6.1 Negative is different from amplification failure. There are various causes of amplification failure:

Whether the main technical elements such as primers, enzymes, buffers, and dntps have been added, whether each component has failed, whether the reaction volume is accurate, whether the DNA has denatured, and whether the cleavage is sufficient, etc.

There are two situations where the amplification reaction may be recorded as a negative result, that is, false negative. One is to detect a certain exogenous specific DNA sequence, such as viruses, bacteria, mycoplasma, etc.

When microorganisms have gene deletions, there is a deficiency in the prompt that their reactions fail and misunderstand errors. Secondly, using 3 '-terminal mismatched allele-specific primers to analyze the mutation of individual bases, amplification failure may indicate that the primers cannot completely anneal with the template. Based on the possible failure, and depending on whether the failed reaction uses mutant primers or normal primers, heterozygotes are mistakenly identified as homozygotes or normal genes.

6.2 To avoid false negatives, another pair of primers can be used to amplify DNA of different lengths from other regions of the genome as controls.

After the PCR reaction is completed, measuring the size of the control DNA band can indicate whether the amplification was successful, but this cannot rule out the possibility of failure due to technical reasons.

Assuming that a negative control can verify primers but increases the possibility of contamination, and the primer control also raises the cost of PCR and the reaction preparation time, it can be considered as appropriate in practical applications.

Of course, when there is doubt about certain negative results, a PCR test can also be repeated, including the sample processing steps.

The PCR reaction is characterized by its large amplification capacity and extremely high sensitivity, but it is highly prone to contamination, leading to false positives and false negatives.

Always pay attention to the monitoring of pollution, consider whether there is pollution and what causes the pollution, and then take measures to prevent and eliminate pollution to ensure the accuracy of the results.

In the actual operation of real-time fluorescent PCR, it is essential to strictly follow the standard operating procedures, eliminate as many factors as possible that may affect the test results, and ensure the accuracy of the test results.