What are PCR artifacts?

The following are common PCR artifacts: -Primer dimers Primer dimers are formed through self-complementarity at the 3' end of the amplification primers. Primer dimers are suspected if product is produced in a template-free reaction (negative control). To avoid primer dimers, primers shouldn't have complementarity at their 3' ends. -Chimeric PCR products Chimeric PCR products can be caused by [...]

What factors contribute to PCR-introduced mutations?

The following factors can contribute to PCR-introduced mutations: -Unbalanced dNTP concentrations Unequal amounts of the four dNTPs can increase base substitution by as high as eight-fold. Using equal concentrations of the four dNTPs is critical for reducing the error rate of the polymerase. -High enzyme concentration -Long incubation times -Lack of 3'?5' exonuclease activity -Magnesium [...]

What types of mutations can be caused by PCR?

PCR polymerases can introduce different types of mutations, including single-base substitutions, deletions, and insertions. Base substitutions are typically caused by misincorporation of an incorrect dNTP during DNA synthesis. Polymerases may generate mutations at locations where one or more nucleotides are lost or gained. The frequency of this type of mutation can be sequence dependent, and [...]

What is PCR overcycling? How do I know if my product is overcycled?

PCR overcycling is when cycling goes beyond the exponential phase of amplification. Overcycling occurs when the following events take place during PCR: -Depletion of substrates (dNTPs or primers). -The reagents (dNTPs or enzymes) are no longer stable at the denaturation temperature. -The PCR polymerase is inhibited by the product (pyrophosphate, duplex DNA). -Competition for reagents (dNTPs and [...]

What are PCR inhibitors?

Impurities that interfere with PCR amplification are known as PCR inhibitors. PCR inhibitors are present in a large variety of sample types and may lead to decreased PCR sensitivity or even false-negative PCR results. PCR inhibitors may have both inorganic and organic origins (Schrader 2012). 1. Inorganic PCR inhibitors include: -Calcium or other metal ions that compete with magnesium -EDTA [...]