Bio-Rad Precision Melt Analysis™ Software - Manual (Page 6)

Precision Melt Analysis Software Instruction Manual
1
1 HRM Analysis
Read this chapter for information about high resolution melt (HRM) analysis.
• Introduction to HRM analysis (below)
• Guidelines for successful HRM analysis (page 2)
• Dye compatibility (page 3)
• HRM analysis in the literature (page 3)
Introduction to HRM Analysis
Real-time PCR assays using non-specific DNA binding dyes such as SYBR
®
Green generally
include a post-PCR melt curve to confirm a single PCR product has been amplified, or to
detect the possible presence of primer-dimers or other unwanted PCR products.
For melt curve analysis the temperature is gradually increased and fluorescence is monitored
as a function of the temperature. As the temperature rises the fluorophore is released from the
denaturing dsDNA and the fluorescence decreases with a noticeable change in slope at the
melting temperature (T
m
) of the dsDNA, the theoretical temperature at which half the DNA is
double stranded and half the DNA is single stranded. The rate of change is determined by
plotting the negative first regression of relative fluorescence (RFU) versus temperature (-
d(RFU)/dT), yielding visible peaks that represents the T
m
of the double-stranded DNA
complexes. Primer-dimers typically melt at lower temperatures due to their smaller size,
enabling primer-dimers or other non-specific products to be discontinued from the amplified
DNA product.
HRM analysis can be considered the next generation of the melt curve technique. HRM
analysis generates DNA melt curve profiles that are both specific and sensitive enough to
distinguish nucleic acid species based on small nucleic acid differences enabling mutation
scanning, methylation analysis, and genotyping.
HRM analysis can be used to characterize samples based on sequence length, GC content
and DNA sequence complementarity. For example, HRM analysis can be used to detect single
base sequence variations such as single nucleotide polymorphisms (SNPs) or to discover
unknown genetic mutations. It can also be used to quantitatively detect a small proportion of
variant DNA in a background of wild-type sequence at sensitivities approaching 5%. This
approach can be used, for example, to study somatically acquired mutations or changes in the
methylation state of CpG islands.