Genotyping of single-nucleotide polymorphisms by high-resolution melting of small amplicons.
BACKGROUND
High-resolution melting of PCR amplicons with the DNA dye LCGreen I was recently introduced as a homogeneous, closed-tube method of genotyping that does not require probes or real-time PCR. We adapted this system to genotype single-nucleotide polymorphisms (SNPs) after rapid-cycle PCR (12 min) of small amplicons (</=50 bp).


METHODS
Engineered plasmids were used to study all possible SNP base changes. In addition, clinical protocols for factor V (Leiden) 1691G>A, prothrombin 20210G>A, methylenetetrahydrofolate reductase (MTHFR) 1298A>C, hemochromatosis (HFE) 187C>G, and beta-globin (hemoglobin S) 17A>T were developed. LCGreen I was included in the reaction mixture before PCR, and high-resolution melting was obtained within 2 min after amplification.


RESULTS
In all cases, heterozygotes were easily identified because heteroduplexes altered the shape of the melting curves. Approximately 84% of human SNPs involve a base exchange between A::T and G::C base pairs, and the homozygotes are easily genotyped by melting temperatures (T(m)s) that differ by 0.8-1.4 degrees C. However, in approximately 16% of SNPs, the bases only switch strands and preserve the base pair, producing very small T(m) differences between homozygotes (<0.4 degrees C). Although most of these cases can be genotyped by T(m), one-fourth (4% of total SNPs) show nearest-neighbor symmetry, and, as predicted, the homozygotes cannot be resolved from each other. In these cases, adding 15% of a known homozygous genotype to unknown samples allows melting curve separation of all three genotypes. This approach was used for the HFE 187C>G protocol, but, as predicted from the sequence changes, was not needed for the other four clinical protocols.


CONCLUSIONS
SNP genotyping by high-resolution melting analysis is simple, rapid, and inexpensive, requiring only PCR, a DNA dye, and melting instrumentation. The method is closed-tube, performed without probes or real-time PCR, and can be completed in less than 2 min after completion of PCR.
Liew
Michael
M
Institute for Clinical and Experimental Pathology, ARUP, Salt Lake City, UT, USA.
Pryor
Robert
R
Palais
Robert
R
Meadows
Cindy
C
Erali
Maria
M
Lyon
Elaine
E
Wittwer
Carl
C
D005165:Factor V / Q000235:genetics; D005838:Genotype; D006432:Hemochromatosis / Q000235:genetics; D006801:Humans; D042965:Methylenetetrahydrofolate Reductase (NADPH2) / Q000235:genetics; D009154:Mutation; D016133:Polymerase Chain Reaction; D020641:Polymorphism, Single Nucleotide; D011516:Prothrombin / Q000235:genetics
D005165:Factor V; D011516:Prothrombin; D042965:Methylenetetrahydrofolate Reductase (NADPH2)