High-Resolution Melting PCR to Determine Sequence Variations in Mutant DNA Sequences

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 site may denature first, releasing the dye. As the remaining dye-binding sites are saturated, the dye fails to re-intercalate, reducing fluorescence intensity. Further, dsDNA dissociates into ssDNA upon incremental heating.

Identify the melting temperature, at which half the dsDNA denatures to ssDNA with a sharp fluorescence decrease. A shift in the melt curve of the amplified DNA fragments identifies the sample's nucleotide sequence variations.

To perform the HRM-PCR protocol, first, dilute the DNA samples in 1.5-milliliter tubes with water to a concentration of 10 nanograms per microliter. Next, dilute the thawed primer solutions in 1.5-milliliter tubes with water to the same concentration of 6 micromolar. Thaw the HRM-PCR kit solutions, and mix carefully by vortexing to ensure the recovery of all contents.

Briefly spin the three vials containing enzymatic mixture with DNA-binding dye, magnesium chloride, and water in a microcentrifuge before opening them. In a 1.5-milliliter tube at room temperature, use the components to prepare the PCR mix for 120-microliter reaction, as described in the text protocol. Mix carefully by vortexing.

It is critical that you work with attention, navigation, according to good practice in molecular biology.

Pipette 19 microliters of the PCR mix into each well of a white multiwell plate. Add 1 microliter of the concentration-adjusted DNA template, then, seal the white multiwell plate with sealing foil. Centrifuge the white multiwell plate for 1 minute at 1500 times g in a standard swing-bucket centrifuge containing a rotor for multiwell plates with suitable adapters. Load the white multiwell plate into the HRM-PCR instrument. Start the HRM-PCR program with the PCR conditions found in the text protocol.

Following the HRM-PCR reaction, perform HRM analysis to determine the CR1 length polymorphism, as described in the text protocol.