High-resolution genotyping by amplicon melting analysis using LCGreen.
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), beta-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 beta-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.
Wittwer
Carl T
CT
Department of Pathology, University of Utah Medical School, Salt Lake City, UT 84132, USA. carl.wittwer@path.utah.edu
Reed
Gudrun H
GH
Gundry
Cameron N
CN
Vandersteen
Joshua G
JG
Pryor
Robert J
RJ
D052160:Benzothiazoles; D001798:Blood Proteins / Q000235:genetics; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D004252:DNA Mutational Analysis / Q000379:methods*; D003959:Diamines; D005456:Fluorescent Dyes; D005838:Genotype; D006444:Hemoglobin C / Q000235:genetics; D006451:Hemoglobin, Sickle / Q000235:genetics; D020180:Heteroduplex Analysis; D006801:Humans; D009930:Organic Chemicals; D016133:Polymerase Chain Reaction; D020641:Polymorphism, Single Nucleotide; D011804:Quinolines; D044402:Receptor, Serotonin, 5-HT2A; D011985:Receptors, Serotonin / Q000235:genetics
D052160:Benzothiazoles; D001798:Blood Proteins; C505032:CFTR protein, human; D003959:Diamines; D005456:Fluorescent Dyes; D006451:Hemoglobin, Sickle; D009930:Organic Chemicals; D011804:Quinolines; D044402:Receptor, Serotonin, 5-HT2A; D011985:Receptors, Serotonin; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; C098022:SYBR Green I; D006444:Hemoglobin C