The primer melting temperature (Tm) should be the same for all primers used in the multiplex reaction. Plan to amplify a 75–200 bp product. Short PCR products are typically amplified with higher ...

Primers need to be aligned to annotated assemblies of the genome of interest (template DNA of LAM-PCR) to avoid unspecific hybridization of the primers.

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If so, the PCR primer would preferentially anneal to and amplify the chromosome with the perfectly matched allele.