Melt-Curve Analysis and Epigenetic Detection

Melt curves

After amplification, the instrument slowly heats the reaction while reading fluorescence from a dye (or probe) that signals double-stranded DNA. As each product melts, fluorescence drops ¹. Plotting the negative derivative (-dF/dT) against temperature turns each melting step into a peak at that product’s Tm ².

• One sharp peak: one specific product.
• An extra, lower peak: primer dimers or nonspecific product.
• A shifted peak: a different sequence - longer, more GC-rich, or with a variant.

This is the standard check on every SYBR Green qPCR run (see Real-Time PCR).

High-resolution melt

High-resolution melt (HRM) uses saturating dyes that fill all double-stranded DNA without inhibiting PCR, and instruments that heat in tiny steps with precise optics ¹. That reveals subtle curve differences:

• Homozygous variants shift the curve slightly (a single base change can move Tm by a fraction of a degree).
• Heterozygotes form heteroduplexes - strands from different alleles paired with a mismatch - that melt earlier, changing the curve’s shape, which is easy to see.
• Uses: mutation scanning (screening an exon for any change before sequencing only the abnormal samples), genotyping known SNPs, and methylation analysis after bisulfite.

Short amplicons (under about 150 bp) maximize sensitivity. Sample DNA must be uniform in quality and salt, since both shift melting.

Probe melts: FRET hybridization probes or beacons, left intact after PCR, melt off a mismatched allele at a lower temperature, calling genotypes with high confidence (see Probe Chemistries).

Detecting methylation

DNA methylation at CpG sites silences genes without changing their sequence ³. Ways to measure it ¹:

Clinical uses: MGMT methylation in glioblastoma, MLH1 promoter methylation in sporadic MSI tumors, imprinting disorders at 15q11-q13 (Prader-Willi, Angelman) and 11p15 (Beckwith-Wiedemann, Silver-Russell), fragile X methylation status, and methylation profiling of brain tumors.

What to take from this

Melt analysis heats product while reading fluorescence; the derivative turns each product into a peak at its Tm, so one peak means one product. HRM with saturating dyes sees heteroduplex shape changes for mutation scanning and genotyping, and intact probes can be melted to call genotypes. Methylation is read after bisulfite conversion, by methylation-sensitive restriction (HpaII vs MspI) or MS-MLPA, natively by long reads, or genome-wide on arrays.