Polymerase chain reaction (PCR) is a widely used technique in molecular biology. It allows researchers to amplify specific DNA fragments from complex mixtures, providing a powerful tool for a wide range of applications from genetic research to medical diagnostics. In addition to standard PCR amplification, variants of this technique have also been developed. These include reverse transcription PCR (RT-PCR), quantitative real-time PCR (qPCR) and digital droplet PCR (ddPCR). Although widely adopted and routinely used, errors can occur leading to poor quality PCR products and thus unreliable results. Proper PCR protocol design and optimization can help reduce errors. An understanding of PCR principles, awareness of common mistakes, and experience can help researchers prevent problems in the first place and troubleshoot problems when they do occur. In this article, we provide a PCR troubleshooting guide covering some of the most common problems researchers encounter and how to overcome them.
1. Low or no PCR product yield
| Causes | Solutions |
|---|---|
| PCR reagents were omitted | Repeat the PCR reaction but check to make sure all components were added. |
| Poor primer design | Repeat primer design process, double check primer specificity, try increasing primer length, or use a different design method. Search literature or databases for effective primers that can amplify the expected DNA fragment. |
| Insufficient primers | Confirm that the primer reserves are prepared correctly. Repeat the test by varying the concentration of the primers. The usual range is 0.05 to 1 μM. |
| Poor template quality | Analyze the plasmid by gel electrophoresis, and analyze the plasmid and gDNA with the A260/280 ratio of the nanopipette to assess its quality. Template DNA was further purified. |
| Complex templates | Unless special polymerases are used, amplification from GC-rich fragments or longer templates may be suboptimal. Template concentrations varied for amplification between plasmids (1 pg 10 ng per 50 μL reaction) and gDNA (1 ng-1 μg per 50 μL reaction). |
| Reaction mix components are compromised | Check expiration date of components. Aliquot biological components of reaction mixture and avoid multiple freeze-thaw cycles. |
| Insufficient cycles | Run a test PCR reaction with different cycles to determine the ideal amount. |
| Insufficient extension time | If the polymerase is not given enough time to amplify the DNA, then there will be no product. Check the manufacturer's polymerase rate and calculate an extension time long enough to allow the DNA to amplify. |
| Incorrect PCR program | Check that the correct program was used, or that the correct program has not been inadvertently changed. Verify the PCR program and repeat the PCR reaction. |
| Incorrect annealing temperature | Check the expected Tm of the designed primers by testing using an annealing temperature gradient. |
| Incorrect blocking temperature | If the blocking temperature is incorrect, the annealing, elongation and melting temperatures will be inaccurate. To correct, perform a heater block calibration. |
2. Incorrect or Non-specific Product
| Causes | Solutions |
|---|---|
| Incorrect primer design | Primers are not designed to amplify the desired DNA fragment, resulting in incorrect product size. Redesign primers to amplify the correct fragment. When non-specific bands appear, increase the primer length to strengthen template binding and minimize false primers. |
| Primers lack specificity | Check the primers for additional complementary regions in the template DNA. |
| Too much primer | Check to make sure the primer stock was prepared correctly. Repeat the test run by varying the primer concentration; a typical range is 0.05-1 μM. |
| Incorrect template concentration | Template concentration varies from plasmid (1pg-10ng per 50μL reaction) to gDNA (1ng-1μg per 50μL reaction). Check the template concentration and adjust the volume of the PCR reaction. Run test reactions to determine optimal concentrations. |
| Suboptimal salt conditions | Magnesium ions can enhance the stability of primer-template binding and increase the affinity of primers to incompletely complementary template sequences. A series of test PCR reactions were performed with different magnesium salt concentrations to determine optimal levels. Also, when thawing the magnesium salt slurry, vortex thoroughly to resuspend any salt that may have precipitated in storage. |
| Annealing temperature too low | Incrementally increase annealing temperature. |
| To replicate prematurely | Prepare the PCR reaction on ice, use a hot-start polymerase, and preheat the PCR machine to denaturing temperature. |
| Contamination by exogenous DNA | Use fresh reagents and work in a dedicated purification area. |
| Nuclease contamination | Repeat the PCR reaction using fresh reagents. |
3. Sequence Error
| Causes | Solutions |
|---|---|
| Low-fidelity polymerase | Use high-fidelity polymerase. |
| Too many cycles | Unnecessary cycles increase the possibility of polymerase errors. Run test reactions to determine the minimum number of cycles required. |
| Unfavorable salt conditions | Reduce the concentration of magnesium salt in the PCR reaction. |
| Degraded or unbalanced dNTPs concentration | Aliquot dNTP stocks to reduce degradation from freeze-thaw cycles; ensure stocks contain equal amounts of dATP, dCTP, dGTP, and dTTP. The dNTP content of the PCR reaction may be too high, test the reaction conditions with a lower concentration. |
| Poor template quality | Template may have been sheared or nicked during purification, or damaged by UV exposure. Prepare fresh template and repeat the PCR reaction. |
| Degraded PCR product | The PCR product may be damaged if it is imaged under UV light before the band is excised in the gel. Repeat the PCR reaction, but limit UV exposure and work faster to excise the bands. |