DOI: 10.1373/49.6.853. High-Resolution Genotyping by Amplicon Melting Analysis Using LCGreen

High-Resolution Genotyping by Amplicon Melting Analysis Using LCGreen

10.1373/49.6.853

Crossref journal-article

Oxford University Press (OUP)

Clinical Chemistry (286)

Abstract

AbstractBackground: 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), β-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 β-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.

Bibliography

Wittwer, C. T., Reed, G. H., Gundry, C. N., Vandersteen, J. G., & Pryor, R. J. (2003). High-Resolution Genotyping by Amplicon Melting Analysis Using LCGreen. Clinical Chemistry, 49(6), 853â860.

Authors 5

Carl T Wittwer (first)
Gudrun H Reed (first)
Cameron N Gundry (first)
Joshua G Vandersteen (first)
Robert J Pryor (first)

References 21 Referenced 1,004

10.1006/abio.1996.9916 / Anal Biochem (1997)
10.1093/clinchem/43.12.2262 / Clin Chem (1997)
{'key': '2020030122351075500_R3', 'first-page': '130–1', 'volume': '22', 'year': '1997', 'journal-title': 'Biotechniques'} / Biotechniques (1997)
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{'key': '2020030122351075500_R7', 'first-page': '258', 'year': '2002', 'journal-title': 'Rapid cycle real-time PCR: methods and applications—microbiology and food analysis'} / Rapid cycle real-time PCR: methods and applications—microbiology and food analysis (2002)
{'key': '2020030122351075500_R8', 'first-page': '180', 'year': '2002', 'journal-title': 'Rapid cycle real-time PCR: methods and applications—genetics and oncology'} / Rapid cycle real-time PCR: methods and applications—genetics and oncology (2002)
10.1006/abio.2000.4957 / Anal Biochem (2001)
10.1373/49.3.396 / Clin Chem (2003)
10.1093/clinchem/47.4.635 / Clin Chem (2001)
10.1016/S0002-9440(10)61760-9 / Am J Pathol (2001)
10.1089/gte.1999.3.365 / Genet Test (1999)
10.1093/clinchem/46.3.425 / Clin Chem (2000)
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Dates

Type	When
Created	21 years, 6 months ago (Feb. 12, 2004, 7:44 p.m.)
Deposited	4 years, 2 months ago (June 14, 2021, 2:33 p.m.)
Indexed	4 days, 14 hours ago (Aug. 28, 2025, 8:40 a.m.)
Issued	22 years, 3 months ago (June 1, 2003)
Published	22 years, 3 months ago (June 1, 2003)
Published Online	22 years, 3 months ago (June 1, 2003)
Published Print	22 years, 3 months ago (June 1, 2003)

Funders 0

None

BibTeX

@article{Wittwer_2003, title={High-Resolution Genotyping by Amplicon Melting Analysis Using LCGreen}, volume={49}, ISSN={1530-8561}, url={http://dx.doi.org/10.1373/49.6.853}, DOI={10.1373/49.6.853}, number={6}, journal={Clinical Chemistry}, publisher={Oxford University Press (OUP)}, author={Wittwer, Carl T and Reed, Gudrun H and Gundry, Cameron N and Vandersteen, Joshua G and Pryor, Robert J}, year={2003}, month=jun, pages={853–860} }

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  "abstract": "<jats:title>Abstract</jats:title><jats:p>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\u2013406). 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.</jats:p><jats:p>Methods: We studied polymorphisms in the hydroxytryptamine receptor 2A (HTR2A) gene (T102C), \u03b2-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.</jats:p><jats:p>Results: The six common \u03b2-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\u20132 min, amplification and analysis were achieved in 10\u201320 min with rapid cycling conditions.</jats:p><jats:p>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.</jats:p>",
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