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