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

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.