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Differentiation

Determination of genetic polymorphisms of the amplified pathogen sequences allows typing of pathogens or detection of mutations associated with genetic diseases.

  • The melting curve of amplification product (upper curves in the melting graph) indicates the temperature at which the probes separate from the amplification product.
  • The peak of the negative first derivative (lower curves in the melting graph) of the melting curve identifies the temperature at which maximum probe separation occurs = melting point (Tm).
  • Single base mismatches reduce the melting point by ~2°C and allow for reliable detection and typing of mutations.

Melting point determination of the amplification products of Babesia gibsoni and Babesia canis.  The Babesia gibsoni/canis real-time PCR assay amplifies the target sequence of both species, but the sequences to which the green fluorescent donor probe attaches differ by 2 bases between B. gibsoni and B. canis.  These differences result in a Tm of 64.2°C for B. gibsoni and of 60.0 °C for B. canis and allow clear identification of each species by melting curve analysis in the same PCR.

Auburn University | College of Veterinary Medicine | Auburn, Alabama 36849 | (334) 844-4546
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