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Mass Spectrometry-Based Loss of Heterozygosity Analysis of Single-Nucleotide Polymorphism Loci in Paraffin Embedded Tumors Using the MassEXTEND Assay 

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Presentation on theme: "Mass Spectrometry-Based Loss of Heterozygosity Analysis of Single-Nucleotide Polymorphism Loci in Paraffin Embedded Tumors Using the MassEXTEND Assay "— Presentation transcript:

1 Mass Spectrometry-Based Loss of Heterozygosity Analysis of Single-Nucleotide Polymorphism Loci in Paraffin Embedded Tumors Using the MassEXTEND Assay  Marjo van Puijenbroek, Jan Willem F. Dierssen, Patrick Stanssens, Ronald van Eijk, Anne Marie Cleton-Jansen, Tom van Wezel, Hans Morreau  The Journal of Molecular Diagnostics  Volume 7, Issue 5, Pages (November 2005) DOI: /S (10)60596-X Copyright © 2005 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

2 Figure 1 Design of the MassEXTEND genotyping of c.827A>C SNP assay. 1) PCR amplification generated a product including the c.827A>C SNP. 2) MassEXTEND reaction that results in two products with different mass: c.827C allele, d, and c.827A allele, d. The Journal of Molecular Diagnostics 2005 7, DOI: ( /S (10)60596-X) Copyright © 2005 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

3 Figure 2 Mass spectra of the three c.827A>C SNP genotypes (A, A/A; B, C/C; C, A/C) and tumor with loss of the A allele (D). The alleles are indicated with thick horizontal arrows. Pausing and probe peaks are indicated above the graph. The Journal of Molecular Diagnostics 2005 7, DOI: ( /S (10)60596-X) Copyright © 2005 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

4 Figure 3 Sequence analysis of the c.827A>C SNP of PTPRJ of flow-sorted cell populations. Distinct cell populations were flow-sorted from the formalin-fixed paraffin-embedded primary tumor and lymph-node metastasis of case A–F: Primary tumor. A: Keratin positive (K pos.) cells can be clearly identified in the forward scatter versus keratin dot plot, compared with a negative control (C). B: After gating on the K pos. cells, a bimodal DNA histogram can be observed with two dominant cycling populations with a DNA index of 1.7 and 2.6, respectively. D: The Keratin negative (K neg.) cells, comprising inflammatory and stromal cells, revealed an unimodal DNA diploid histogram.17 E and F: Sequence analysis of fraction ID 1.7 and fraction ID 2.6 showed loss of the A allele in both populations. G–L: Lymph-node metastasis. G: Forward scatter versus keratin dot plot. I: Negative control. H: Gating on the K pos. cells shows an unimodal DNA histogram with a DNA index of 2.6. These cells probably branched from the second DNA aneuploid population (DI = 2.6) of the primary tumor. J: Unimodal DNA diploid histogram of the K neg. cells. K: Sequence analysis of ID 2.6 fraction showed loss of the A allele. L: The K neg. cells are diploid and show the normal A/C genotype. The Journal of Molecular Diagnostics 2005 7, DOI: ( /S (10)60596-X) Copyright © 2005 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

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