Several factors impact PCR success. Apart from primer design, the two most important are template quality and polymerase robustness. Harsh sample preparation workflows may damage nucleic acids, thereby reducing the number of available targets for the polymerase enzyme. This can introduce selection bias into the sample, which is then amplified and exaggerated during PCR. As for polymerase robustness, reagents used and not sufficiently removed during sample preparation can inhibit polymerases. Additionally, the enzymes can be prone to misreads that introduce errors.
Fortunately, there are ways to avoid these pitfalls. New technologies such as GenElute™-E purification allow for gentler, streamlined workflows that reduce damage and minimize bias. These workflows are accompanied by superior polymerase enzymes that offer lower error rates and can overcome inhibitory contaminants.
• Common chemical and biological impurities to avoid during nucleic acid extraction
• Strategies for optimizing DNA/RNA quality to improve PCR reaction yield
• Selecting a polymerase for robust PCR assays from crude samples
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