Amplicon DNA Melting Analysis for Mutation Scanning and Genotyping: Cross-Platform Comparison of Instruments and Dyes
作者： Mark G Herrmann , Jacob D Durtschi , L Kathryn Bromley , Carl T Wittwer , Karl V Voelkerding
DOI: 10.1373/CLINCHEM.2005.063438
关键词: Genotyping 、 Molecular biology 、 Melting curve analysis 、 Locus (genetics) 、 Amplicon 、 Genotype 、 Biology 、 SYBR Green I 、 Nucleic acid thermodynamics 、 High Resolution Melt Analysis
摘要: Background: DNA melting analysis for genotyping and mutation scanning of PCR products by use high-resolution instruments with special “saturation” dyes has recently been reported. The comparative performance other not evaluated. Methods: A 110-bp fragment the β-globin gene including sickle cell anemia locus (A17T) was amplified in presence either saturating dye, LCGreen Plus, or SYBR Green I. Amplicons 3 different genotypes (wild-type, heterozygous, homozygous mutants) were melted on 9 (ABI 7000 7900HT, Bio-Rad iCycler, Cepheid SmartCycler, Corbett Rotor-Gene 3000, Idaho Technology HR-1 LightScanner, Roche LightCycler 1.2 2.0) at a rate 0.1 °C/s as recommended manufacturer. ability each instrument/dye combination to genotype temperature ( T m) scan heterozygotes curve shape evaluated. Results: Resolution varied greatly among 15-fold difference m SD (0.018 0.274 °C) 19-fold (LCGreen Plus) 33-fold (SYBR I) signal-to-noise ratio. These factors limit most accurately single-nucleotide polymorphisms amplicon melting. Plate (96-well) showed greatest variance spatial differences across plates. Either I Plus could be used m, but only useful heterozygote scanning. However, an argon laser because spectral mismatch. All compatible able detect altered shape. specifically designed displayed least variation, suggesting better sensitivity specificity. Conclusion: Different vary widely their variants whole-amplicon analysis.
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