JoVE Encyclopedie van Experimenten
Biologische Technieken
0 weergaven • 4:09 min. • July 8th, 2025
High-resolution melting polymerase chain reaction, HRM-PCR, detects double-stranded DNA, dsDNA sequence variations with high precision.
Begin with a master mix containing inactive Taq polymerase, deoxyribonucleotide triphosphates — dNTPs — and the fluorescent reporter dye. Add the forward and reverse primers. Pipette the mixture to a PCR plate's wells.
Add the dsDNA template. Seal the plate, preventing mixture evaporation. Begin the PCR.
The high denaturing temperature denatures dsDNA to single-stranded DNA, ssDNA, and activates the polymerase. The reaction is then cooled to the annealing temperature, facilitating forward and reverse primer binding to ssDNAs via site-specific complementary base pairing.
Activated polymerase gets recruited, extending the DNA-primer duplex by dNTP addition.
The fluorescent reporter dye — a dsDNA-specific dye present in saturating concentrations — intercalates within the entire duplex, fluorescing brightly. The duplex undergoes multiple PCR cycles, producing several variant-containing fragments.
Post-PCR, perform melt curve analysis by re-rising the reaction temperature steadily over time.
Depending on the nucleotide sequence, the variant-containing s
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