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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.