Expanded Instrument Comparison of Amplicon DNA Melting Analysis for Mutation Scanning and Genotyping
Open Access
- 1 August 2007
- journal article
- Published by Oxford University Press (OUP) in Clinical Chemistry
- Vol. 53 (8), 1544-1548
- https://doi.org/10.1373/clinchem.2007.088120
Abstract
Background: Additional instruments have become available since instruments for DNA melting analysis of PCR products for genotyping and mutation scanning were compared. We assessed the performance of these new instruments for genotyping and scanning for mutations. Methods: A 110-bp fragment of the β-globin gene including the sickle cell anemia locus (HBB c. 20A>T) was amplified by PCR in the presence of LCGreen Plus or SYBR Green I. Amplicons of 4 different genotypes [wild-type, homozygous, and heterozygous HBB c. 20A>T and double-heterozygote HBB c. (9C>T; 20A>T)] were melted on 7 different instruments [Applied Biosystems 7300, Corbett Life Sciences Rotor-Gene 6500HRM, Eppendorf Mastercycler RealPlex4S, Idaho Technology LightScanner (384 well), Roche LightCycler 480 (96 and 384 well) and Stratagene Mx3005p] at a rate of 0.61 °C/s or when this was not possible, at 0.50 °C steps. We evaluated the ability of each instrument to genotype by melting temperature (Tm) and to scan for heterozygotes by curve shape. Results: The ability of most instruments to accurately genotype single-base changes by amplicon melting was limited by spatial temperature variation across the plate (SD of Tm = 0.020 to 0.264 °C). Other variables such as data density, signal-to-noise ratio, and melting rate also affected heterozygote scanning. Conclusions: Different instruments vary widely in their ability to genotype homozygous variants and scan for heterozygotes by whole amplicon melting analysis. Instruments specifically designed for high-resolution melting, however, displayed the least variation, suggesting better genotyping accuracy and scanning sensitivity and specificity.Keywords
Funding Information
- Applied Biosystems
- Corbett Life Science
- Eppendorf
- Idaho Technology
- ARUP Laboratories
This publication has 14 references indexed in Scilit:
- Instrument Comparison for Heterozygote Scanning of Single and Double Heterozygotes: A Correction and Extension of Herrmann et al., Clin Chem 2006;52:494-503Clinical Chemistry, 2007
- High resolution melting analysis for the rapid and sensitive detection of mutations in clinical samples: KRAS codon 12 and 13 mutations in non-small cell lung cancerBMC Cancer, 2006
- Amplicon DNA Melting Analysis for Mutation Scanning and Genotyping: Cross-Platform Comparison of Instruments and DyesClinical Chemistry, 2006
- Quantitative heteroduplex analysis for single nucleotide polymorphism genotypingAnalytical Biochemistry, 2005
- Distinguishing Different DNA Heterozygotes by High-Resolution MeltingClinical Chemistry, 2005
- Sensitivity and Specificity of Single-Nucleotide Polymorphism Scanning by High-Resolution Melting AnalysisClinical Chemistry, 2004
- Genotyping of Single-Nucleotide Polymorphisms by High-Resolution Melting of Small AmpliconsClinical Chemistry, 2004
- High‐resolution DNA melting curve analysis to establish HLA genotypic identityTissue Antigens, 2004
- High-Resolution Genotyping by Amplicon Melting Analysis Using LCGreenClinical Chemistry, 2003
- Amplicon Melting Analysis with Labeled Primers: A Closed-Tube Method for Differentiating Homozygotes and HeterozygotesClinical Chemistry, 2003