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For What Applications is Fidelity Important?

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Fidelity is important for applications in which the DNA sequence must be correct after amplification, including:

  • Cloning/subcloning from in vitro amplified material (PCR, WGA, etc.) for protein expression or gene studies
  • SNP analysis by cloning and sequencing
  • RNA analysis by RT-PCR
  • Applications that involve sequencing of in vitro amplified material

Fidelity is less important if the PCR amplified product is directly sequenced by Sanger sequencing (without an intervening cloning step), where the signal is an average of the input amplicons. However, it is more important for next generation and single molecule sequencing techniques. Fidelity is also less important for those diagnostic applications in which sequencing is not required after amplification, and the read-out is the presence or absence of a product.

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