Characterization of Aberrant Melting Peaks in Unlabeled Probe Assays

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Presentation transcript:

Characterization of Aberrant Melting Peaks in Unlabeled Probe Assays Shale Dames, Rebecca L. Margraf, David C. Pattison, Carl T. Wittwer, Karl V. Voelkerding The Journal of Molecular Diagnostics Volume 9, Issue 3, Pages 290-296 (July 2007) DOI: 10.2353/jmoldx.2007.060139 Copyright © 2007 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 1 Unlabeled probe melting analysis of exon 10 of the RET gene. A: High-resolution melting profile observed with 3′ phosphate blocked unlabeled probe (probe-phos). B: Diagram of amplification products expected using primers 10F, 10R, and incompletely blocked probe-phos. C: Unlabeled probe reaction visualized on a 4% agarose gel. Products include 146-bp dsDNA using primers 10F and 10R (band 1), 146-nucleotide ssDNA (band 2), 62-bp amplicon derived from extension of unblocked probe-phos and 10R (band 3), and 62-nucleotide ssDNA (band 4). Lane Phos used probe-phos and is represented by the melting profile shown in A. Lane No block was run with probe-OH and 10R primers to demonstrate size differences between the 62-nucleotide ssDNA and 62-bp dsDNA amplicon (asterisk). The asterisk denotes the 62-bp probe-phos and 10R amplicon. D: Sequence of cloned unlabeled probe products. Sequence of 146-bp wild-type amplicon 10F and 10R (band 1, C) and the 62-bp probe-phos and 10R-derived amplicon (band 3, C). The amplification products were isolated from the same unlabeled probe reaction, demonstrating that the 62-bp amplicon was derived by extension of unblocked probe-phos. The Journal of Molecular Diagnostics 2007 9, 290-296DOI: (10.2353/jmoldx.2007.060139) Copyright © 2007 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 2 Day 0 high-resolution melting analysis of blocked probes. A: Probes blocked with C3, C6, and InvT provide the least amount of DNA polymerase extension on day 0, as shown by the heights of peaks 2 and 3. Probe-phos extension is higher as a new oligonucleotide and shown in red for comparison. B: Probe-mis, phos, C6, and probe-OH are shown for comparison. Probe-mis displays aberrant melting profiles and cannot be used for melting analysis. The Journal of Molecular Diagnostics 2007 9, 290-296DOI: (10.2353/jmoldx.2007.060139) Copyright © 2007 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 3 Estimation of blocking stability after 50 freeze-thaw cycles. A: Complete melting profile of unblocked to blocked probes representing 0, 1, 2, and 5% unblocked. B: Melting profiles of C3, RevT, and C6 compared with unblocked percentage. The best blocking stability is amino C6, followed by InvT, and C3. C: Melting profile of phosphate compared with unblocked percentages shows that phosphate is the least stable of all blocks tested. The Journal of Molecular Diagnostics 2007 9, 290-296DOI: (10.2353/jmoldx.2007.060139) Copyright © 2007 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions