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1. HRM-PCR Protocol
<ol>
	<li>Thaw the DNA samples. Dilute the DNA samples in 1.5 mL tubes with water to adjust them to a concentration of 10 ng/&#181;L 
	NOTE: The total volume of diluted DNA should be between 2 &#181;L and 10 &#181;L.</li>
	<li>Thaw the primer solutions. Dilute the primer solutions in 1.5-mL tubes with water to adjust them to the same concentration of 6 &#181;M. 
	NOTE: The primer sequences and reaction conditions are provided in Table 1.</...

high-resolution melting pcr to determine sequence variations in mutant dna sequences

Source: Kisserli, A., et al. <a href="https://app.jove.com/v/56012">High-resolution Melting PCR for Complement Receptor 1 Length Polymorphism Genotyping: An Innovative Tool for Alzheimer&#39;s Disease Gene Susceptibility Assessment.</a> J. Vis. Exp. (2017).
In this video, we describe a high-resolution melting PCR technique to determine the length polymorphism of DNA fragments of varying sequence lengths, based on melting temperature analysis.

<img alt="Figure 1" src="/files/ftp_upload/21360/21360_Figure2.jpg" /> 
Figure 1: Screenshots of the graphical interface of the software used in step 2 of the protocol. (A) Open the gene scanning software. (B) Amplification program and melting curve program. (C) Click on Sample editor in the Module bar. (D) Select Scanning. (E) Define the properties of the samples. ...

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

Source: Kisserli, A., et al. High-resolution Melting PCR for Complement Receptor 1 Length Polymorphism Genotyping: An Innovative Tool for Alzheimer&#39;s ...

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 &#8212; dNTPs &#8212; and the fluorescent reporter dye. Add the forward and reverse primers. Pipette the mixture to a PCR plate&#39;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 &#8212; a dsDNA-specific dye present in saturating concentrations &#8212; 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&#39;s nucleotide sequence variations.

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

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