Kathleen M. Murphy, Tanya Geiger, Michael J. Hafez, James R

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Presentation transcript:

A Single Nucleotide Primer Extension Assay to Detect the APC I1307K Gene Variant Kathleen M. Murphy, Tanya Geiger, Michael J. Hafez, James R. Eshleman, Constance A. Griffin, Karin D. Berg The Journal of Molecular Diagnostics Volume 5, Issue 4, Pages 222-226 (November 2003) DOI: 10.1016/S1525-1578(10)60477-1 Copyright © 2003 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 1 Schematic of the single nucleotide primer extension assay. The boxes represent the PCR product, the sequence surrounding the I1307K variant is demonstrated. The forward (21-base oligomer) and reverse (24-base oligomer) SNaPshot primers are indicated by arrows. A wild-type and mutant allele are depicted demonstrating the fluorescent ddNTP which extends each primer. The Journal of Molecular Diagnostics 2003 5, 222-226DOI: (10.1016/S1525-1578(10)60477-1) Copyright © 2003 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 2 Examples of SNaPshot results. Capillary electrophoresis pherograms, x axis is size in bases, y axis is fluorescence intensity. The orange peaks represent the internal size standard. The arrows labeled “F” indicate SNaPshot products resulting from extension of the forward primer, sizing at 26 bases. The arrows labeled “R” indicate SNaPshot products resulting from extension of the reverse primer, sizing at either 30 or 31 bases. The addition of a thymidine (T) ddNTP to a primer results in a red-colored product/peak, the addition of an adenine (A) ddNTP results in a green-colored peak. A: Wild-type result demonstrating a red (T) peak at 26 bases resulting from extension of the forward primer and a green (A) peak at 30 bases resulting from extension of the reverse primer. B: Heterozygous mutant result demonstrating superimposed green (A) and red (T) peaks at 26 bases resulting from extension of the forward primer. The green (A) peak at 30 bases and the red (T) peak at 31 bases result from extension of the reverse primer. The green and red reverse primer peaks travel at different sizes due to the effect of the fluorophore on migration. C: Homozygous mutant result demonstrating a green (A) peak at 26 bases resulting from extension of the forward primer and a red peak at 31 bases resulting from extension of the reverse primer. The Journal of Molecular Diagnostics 2003 5, 222-226DOI: (10.1016/S1525-1578(10)60477-1) Copyright © 2003 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 3 Results from the ASO assay. PCR products were slot-blotted onto two nylon membranes and probed with either a wild-type or mutant radioactive probe. After incubation and washing, the membranes were exposed to film and the results were compared. Lane 1 is a no template control. Lanes 2, 4,and 5 hybridized with both the wild-type and mutant probes and are thus interpreted as heterozygous positive for the APC I1307K variant. Lanes 3 and 6 hybridized with the wild-type probe only and are thus interpreted as wild-type (negative for the I1307K variant). The Journal of Molecular Diagnostics 2003 5, 222-226DOI: (10.1016/S1525-1578(10)60477-1) Copyright © 2003 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions