How do you troubleshoot sample preparation issues in multiplex PCR?

1 Primer design

One of the key factors for successful multiplex PCR is the design of primers that are specific, compatible, and balanced. Specific primers avoid non-specific amplification and cross-reactivity with other targets or genomic DNA. Compatible primers have similar melting temperatures, annealing times, and GC contents, and do not form dimers or secondary structures. Balanced primers have similar concentrations and lengths, and generate products with similar sizes and yields. To design primers for multiplex PCR, you can use online tools, such as Primer3Plus or Primer-BLAST, that allow you to check for these criteria and optimize your primer sets.

2 Template quality

Another important factor for successful multiplex PCR is the quality of the template DNA or RNA. Template quality can affect the efficiency, specificity, and sensitivity of the amplification. Poor template quality can result from degradation, contamination, or inhibition of the template or the polymerase. To ensure good template quality, you should use appropriate extraction methods, storage conditions, and quality control measures, such as spectrophotometry, gel electrophoresis, or qPCR. You should also use the optimal amount of template for your multiplex PCR, depending on the target and the primer design.

3 Reaction optimization

A third factor for successful multiplex PCR is the optimization of the reaction conditions, such as the buffer, the polymerase, the magnesium, the dNTPs, the primers, and the cycling parameters. Reaction optimization can improve the specificity, efficiency, and reproducibility of the multiplex PCR. However, it can also be time-consuming and costly, especially for high-throughput applications. To optimize your multiplex PCR reaction, you can use a systematic approach, such as a factorial design or a gradient PCR, that allows you to test different variables and find the optimal combination. You can also use commercial kits or master mixes that are designed for multiplex PCR and provide standardized conditions.

4 Troubleshooting tips

Even with careful sample preparation, multiplex PCR can sometimes fail or produce unexpected results. Some common problems, their possible causes, and solutions include no amplification or low yield, which may be due to insufficient or degraded template, incorrect primer concentration or annealing temperature, insufficient polymerase or magnesium, or inhibition of the reaction. To fix this issue, you can increase the template amount or quality, adjust the primer concentration or annealing temperature, add more polymerase or magnesium, or use a different buffer or extraction method. Non-specific amplification or primer dimers may be caused by unspecific or incompatible primers, too high primer concentration or annealing temperature, too long extension time, or too many cycles. To resolve this problem, you can redesign the primers or use a different primer set, reduce the primer concentration or annealing temperature, shorten the extension time, or reduce the number of cycles. Unequal amplification or preferential amplification may be due to unbalanced or incompatible primers, too low primer concentration or annealing temperature, too short extension time, or too few cycles. To address this issue you can balance the primers or use a different primer set, increase the primer concentration or annealing temperature, lengthen the extension time, or increase the number of cycles. Smearing or multiple bands may result from unspecific or incompatible primers, too high template amount or quality, too high polymerase or magnesium, and too long extension time. To fix this problem you can redesign the primers or use a different primer set, reduce the template amount or quality, reduce the polymerase or magnesium and shorten the extension time.

5 Best practices

To ensure successful multiplex PCR, it is essential to follow some best practices. These include using a positive and negative control to verify specificity and sensitivity, a reference gene or internal control to normalize results, a validation kit or standard curve to assess efficiency and linearity, a gel documentation system or capillary electrophoresis system to analyze products and confirm sizes and quantities, and good laboratory practice with aseptic technique to prevent contamination and cross-talk. With careful sample preparation and optimization, multiplex PCR can be a valuable tool for molecular diagnostics and quality control. Following these tips can help you troubleshoot sample preparation issues in multiplex PCR and obtain reliable and accurate results.

6 Here’s what else to consider

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