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Genotyping DNA Variants with High-Resolution Melting Analysis

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Genotyping

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1492))

Abstract

High-resolution melting analysis (HRMA) is a simple, quick, and effective method to scan and screen PCR amplicons for sequence variants. HRMA is a nondestructive closed tube assay; after PCR, DNA melting can directly be performed on the amplified samples without any purification or separation steps. For single SNP genotyping, HRMA is an attractive alternative to Sanger sequencing, restriction enzyme analysis, and hydrolysis probes.

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References

  1. Zhou L, Wang L, Palais R et al (2005) High-resolution DNA melting analysis for simultaneous mutation scanning and genotyping in solution. Clin Chem 51:1770–1777

    Article  CAS  PubMed  Google Scholar 

  2. Erali M, Voelkerding KV, Wittwer CT (2008) High resolution melting applications for clinical laboratory medicine. Exp Mol Pathol 85:50–58

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Reed GH, Wittwer CT (2004) Sensitivity and specificity of single-nucleotide polymorphism scanning by high-resolution melting analysis. Clin Chem 50:1748–1754

    Article  CAS  PubMed  Google Scholar 

  4. Zhou L, Myers AN, Vandersteen JG et al (2004) Closed-tube genotyping with unlabeled oligonucleotide probes and a saturating DNA Dye. Clin Chem 50:1328–1335

    Article  CAS  PubMed  Google Scholar 

  5. Cradic KW, Wells JE, Allen L et al (2004) Substitution of 3′-phosphate Cap with a carbon-based blocker reduces the possibility of fluorescence resonance energy transfer probe failure in real-time PCR. Clin Chem 50:1080–1082

    Article  CAS  PubMed  Google Scholar 

  6. Vossen RHAM, Duijn M, Daha MR et al (2010) High-throughput genotyping of mannose-binding lectin variants using high-resolution DNA-melting analysis. Hum Mutat 31:E186–E193

    Article  CAS  Google Scholar 

  7. Liew M, Pryor R, Palais R et al (2004) Genotyping of single-nucleotide polymorphisms by high-resolution melting of small amplicons. Clin Chem 50:1156–1164

    Article  CAS  PubMed  Google Scholar 

  8. Gundry CN, Dobrowolski SF et al (2008) Base-pair neutral Homozygotes can be discriminated by calibrated high-resolution melting of small amplicons. Nucleic Acids Res 36:3401–3408

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Herrmann MG, Durtschi JD, Wittwer CT, Voelkerding KV (2007) Expanded instrument comparison of amplicon DNA melting analysis for mutation scanning and genotyping. Clin Chem 53:1544–1548

    Article  CAS  PubMed  Google Scholar 

  10. Eijk R, Puijenbroek M, Chhatta AR et al (2010) Sensitive and specific KRAS somatic mutation analysis on whole-genome amplified DNA from archival tissues. J Mol Diagn 12:28–34

    Google Scholar 

  11. Vossen RHAM, Aten E, Roos A et al (2009) High-resolution melting analysis (HRMA)—more than just sequence variant screening. Hum Mutat 30:860–866

    Article  CAS  PubMed  Google Scholar 

  12. Herrmann MG, Durtschi JD, Bromley LK et al (2006) Amplicon DNA melting analysis for mutation scanning and genotyping: cross-platform comparison of instruments and dyes. Clin Chem 52:494–503

    Article  CAS  PubMed  Google Scholar 

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Correspondence to Rolf H. A. M. Vossen .

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Vossen, R.H.A.M. (2017). Genotyping DNA Variants with High-Resolution Melting Analysis. In: White, S., Cantsilieris, S. (eds) Genotyping. Methods in Molecular Biology, vol 1492. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6442-0_2

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  • DOI: https://doi.org/10.1007/978-1-4939-6442-0_2

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-6440-6

  • Online ISBN: 978-1-4939-6442-0

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