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Transcript
Problem Multiples melting peaks observed for nuclear gene (more than 2) Amplicon melting transitions not visible or are very small Possible reason Multiple melting domains Additional background peaks Secondary PCR products Solutions Cut the fragments according to overcome (or use internal probes). Presence of mutations in Redesign primer outside mutation area. primer sequence Amplicon too long Design primers for shorter amplicon length and flank melt domains. Low PCR yield Optimize PCR to enhance product yield. Optimize PCR conditions to obtain clean product or design new primers without secondary structures. Inconsistent genomic DNA Ensure that the genomic DNA concentration preparation and buffer is consistent. Melting curves shapes didn’t DNA purification methods Ensure that same DNA extraction technique is correspond to the known samples differ between the sample employed. Identification of variants laborious homozygous Low level of mutations in Generate heteroduplexes by adding wild-type samples generated Tms reference to samples DNA before shift <0.5°C. amplification for nuclear gene or increase amplicon size for mitochondrial gene to generate Tms shift>0.5°C.
