Michael T. Seipp, Jacob D. Durtschi, Michael A

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Presentation transcript:

Unlabeled Oligonucleotides as Internal Temperature Controls for Genotyping by Amplicon Melting Michael T. Seipp, Jacob D. Durtschi, Michael A. Liew, Jamie Williams, Kristy Damjanovich, Genevieve Pont-Kingdon, Elaine Lyon, Karl V. Voelkerding, Carl T. Wittwer The Journal of Molecular Diagnostics Volume 9, Issue 3, Pages 284-289 (July 2007) DOI: 10.2353/jmoldx.2007.060136 Copyright © 2007 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 1 Derivative melting curves of selected internal temperature controls (Table 1). The 35-bp low-temperature control was combined with the 50-bp high-temperature control (gray curve). The black curve included the 35-bp low-temperature control with a 3-bp deletion on one strand and the 50-bp high-temperature control with eight locked nucleic acid bases on one strand. Both mixtures were melted under PCR conditions in duplicate. The Journal of Molecular Diagnostics 2007 9, 284-289DOI: (10.2353/jmoldx.2007.060136) Copyright © 2007 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 2 Derivative melting plots of MTHFR samples extracted using different methods. Samples were extracted in parallel using the MagNA Pure LC system (thick black line), QIAamp DNA blood mini kit (thin black line), and the Puregene DNA blood kit (dotted black line). The sample genotypes shown were 1298AA/677CC and 1298CC/677TT. A: Derivative melting profiles shown without temperature correction. B: Derivative melting profiles shown with temperature correction. The brackets represent Tm ranges expected for the homozygous MTHFR genotypes. The Journal of Molecular Diagnostics 2007 9, 284-289DOI: (10.2353/jmoldx.2007.060136) Copyright © 2007 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 3 Duplex MTHFR derivative melting plots for genotyping the 1298A>C and 677C>T variants. Genotypes shown are 1298AA/677CC (thick black line), 1298CC/677CC (thin black line), and 1298AA/677TT (gray line). A: Without temperature correction. B: With temperature correction using high- and low-temperature controls (not shown). The Journal of Molecular Diagnostics 2007 9, 284-289DOI: (10.2353/jmoldx.2007.060136) Copyright © 2007 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 4 Genotype distribution and derivative melting plots for 677C>T MTHFR genotyping. Thirty-seven selected DNA samples were blinded and genotyped by the duplex amplicon melting assay. A: Genotype distribution determined by HybProbe analysis. The MTHFR 1298CC/677TT genotype was not available and is presumed lethal.13,23 B: Two controls and the two discordant samples are shown without temperature correction. C: The same samples are shown after temperature correction. The solid black line shows 677TT and the solid gray line shows 677CC genotypes. The dotted black and gray lines marked with the asterisk indicate the samples that required temperature correction for correct genotyping. The Journal of Molecular Diagnostics 2007 9, 284-289DOI: (10.2353/jmoldx.2007.060136) Copyright © 2007 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions