Bio-Rad Precision Melt Analysis™ Software - Manual (Page 7)

HRM Analysis
2
SNP Genotyping
Representative of the smallest genetic change, the detection and genotyping of SNPs
underlines the sensitivity of HRM analysis. Unknown mutations are often a single nucleotide
change, but they may also comprise multiple base changes, insertions and/or deletions. In
general, the more base changes in the DNA, the easier they are to detect by HRM.
SNPs have been divided into four classes as summarized in Table 3, the most difficult to
genotype are the class 4 (A>T conversions).
Table 3. SNP classes as defined by Venter et al. (2001)
While the shift in Tm is important, what enables even small changes in Tm to be analyzed
using HRM is the magnitude of change in the fluorescence intensity (y-axis). This change can
be accentuated by minimizing the amplicon size, as discussed below.
For SNP analysis, homozygous allelic variants are characterized by a temperature (x-axis) shift
in a HRM melt curve, whereas heterozygotes are characterized by a change in melt curve
shape. The change in curve shape is a result of destabilized heteroduplex annealing between
some of the wild type and variant strands. The heterozygote melting curve is thus a composite
of both heteroduplex and homoduplex components, and because it dissociates more readily it
shifts to a lower temperature.
Guidelines for Successful HRM Analysis
The success of HRM analysis highly depends on the quality of the individual PCR product and
the specific sequence under investigation. All experimental parameters must be controlled and
highly reproducible from sample to sample to ensure successful HRM analysis.
Recommended guidelines for successful HRM analysis are provided below.
1. Analyze small DNA amplicons
Analyzing amplicons smaller than 150 bp is preferable, especially when sites with a known
polymorphism are investigated. It is possible to detect sequence variations with longer
amplicons, however, a single base variation influences the melting behavior of a 100 bp
amplicon more than a 600 bp amplicon.
2. Analyze a single pure product
Avoid sequences that are likely to form non-specific products or primer dimers. Always run
BLAST search (http://www.ncbi.nlm.nih.gov/BLAST) to check the specificity of the primers
sequences to the target species and gene. In addition, bad resolution or poor grouping may
occur when secondary structures in single-stranded or partially denatured DNA are present.
The amplicon sequences should be entered into MFOLD (http://mfold.rna.albany.edu/
?q=mfold/dna-folding-form) to assure that they do not form any secondary structures during
PCR.
SNP Class Base Change Typical T
m
Melt Curve Shift Rarity (in the Human Genome)
1 C/T and G/A Large >0.5°C
Very small <0.2°C
64%
2 C/A and G/T 20%
3 C/G 9%
4 A/T 7%