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1 Department of Pathology, University of Utah Medical School, Salt Lake City, UT 84132.
aAuthor for correspondence. Fax 801-581-4517; e-mail carl.wittwer{at}path.utah.edu.
Background: High-resolution amplicon melting analysis was recently introduced as a closed-tube method for genotyping and mutation scanning (Gundry et al. Clin Chem 2003;49:396–406). The technique required a fluorescently labeled primer and was limited to the detection of mutations residing in the melting domain of the labeled primer. Our aim was to develop a closed-tube system for genotyping and mutation scanning that did not require labeled oligonucleotides.
Methods: We studied polymorphisms in the hydroxytryptamine receptor 2A (HTR2A) gene (T102C), ß-globin (hemoglobins S and C) gene, and cystic fibrosis (F508del, F508C, I507del) gene. PCR was performed in the presence of the double-stranded DNA dye LCGreen, and high-resolution amplicon melting curves were obtained. After fluorescence normalization, temperature adjustment, and/or difference analysis, sequence alterations were distinguished by curve shape and/or position. Heterozygous DNA was identified by the low-temperature melting of heteroduplexes not observed with other dyes commonly used in real-time PCR.
Results: The six common ß-globin genotypes (AA, AS, AC, SS, CC, and SC) were all distinguished in a 110-bp amplicon. The HTR2A single-nucleotide polymorphism was genotyped in a 544-bp fragment that split into two melting domains. Because melting curve acquisition required only 1–2 min, amplification and analysis were achieved in 10–20 min with rapid cycling conditions.
Conclusions: High-resolution melting analysis of PCR products amplified in the presence of LCGreen can identify both heterozygous and homozygous sequence variants. The technique requires only the usual unlabeled primers and a generic double-stranded DNA dye added before PCR for amplicon genotyping, and is a promising method for mutation screening.
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M. Liew, R. Pryor, R. Palais, C. Meadows, M. Erali, E. Lyon, and C. Wittwer Genotyping of Single-Nucleotide Polymorphisms by High-Resolution Melting of Small Amplicons Clin. Chem., July 1, 2004; 50(7): 1156 - 1164. [Abstract] [Full Text] [PDF]
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S. Giglio, P. T. Monis, and C. P. Saint Demonstration of preferential binding of SYBR Green I to specific DNA fragments in real-time multiplex PCR Nucleic Acids Res., November 15, 2003; 31(22): e136 - e136. [Abstract] [Full Text] [PDF]
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