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Vanderbilt-Ingram Cancer CenterVanderbilt-Ingram Cancer Center

 

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SNaPshot Genotyping

 

As part of the Vanderbilt-Ingram Cancer Center's Personalized Cancer Medicine Initiative (PCMI) SNaPshot screens for melanoma, non-small cell lung cancer (NSCLC), colon cancer, and breast cance r were developed to prioritize anti-cancer therapy in the clinic (1, 2). These SNaPshot screens are now available to investigators, on a research basis, through the ITR. For more information about the therapeutic implications of these mutations in tumors please visit My Cancer Genome at www.mycancergenome.org.

Click here for SNaPshot screens available (downloadable pdf)

ITR requires only ONE of the following per sample:

  1. 400 ng genomic DNA (quantified by a double-stranded DNA detection assay-e.g. picogreen) or 1 ug genomic DNA (quantified by nanodrop)

  2. FFPE tissue (block OR 4-10 um sections from a 1cm x 1cm tumor)

  3. Frozen cell pellet from > 6 cm dish

SNaPshot genotyping is a fast, high-throughput, multiplex mutational profiling method based on the Applied Biosystems SNaPshot platform (Figure 1) (3). One advantage of SNaPshot genotyping over conventional dideoxy-nucleotide (Sanger) sequencing is that mutations can be detected when mutant DNA comprises as low as 5% of the total DNA. In contrast, Sanger sequencing can only detect mutant alleles when they comprise >20-25% of a sample.

 

 Figure 1. SNaPshot genotyping. The melanoma, NSCLC, colon cancer and breast cancer screens utilize SNaPshot technology, which involves multiplexed amplification of DNA targets by PCR with unlabeled oligonucleotide primers (Multiplex PCR), multiplexed single-base primer extension with fluorescently-labeled dideoxynucleotides (ddNTPs) (Single-Base Extension), and analysis of labeled-primer extension products by capillary electrophoresis.

References

  1. Abramson VG, Cooper Lloyd M, Ballinger T, Sanders ME, Du L, Lai D, Su Z, Mayer I, Levy M, LaFrance DR, Vnencak-Jones CL, Shyr Y, Dahlman KB, Pao W, Arteaga CL. (2014). Characterization of breast cancers with PI3K mutations in an academic practice setting using SNaPshot profiling. Breast Cancer Research and Treatment. 145: 389-399.

  2. Lovely C*, Dahlman KB*, Fohn L*, Su Z*, Dias-Santagata D, Hicks DJ, Hucks D, Berry, E, Terry C, Duke M Su Y, Sobolik-Delmaire T, Richmond A, Kelley MC, Vnencak-Jones CL, Iafrate JA, Sosman J, Pao W (2012). Routine multiple mutational profiling of melanomas enables enrollment in genotype-driven therapeutic trials. PLoS ONE. 7: e35309. *These authors contributed equally.

  3. Su Z, Dias-Santagata D, Duke M, et al. (2011). A platform for rapid detection of multiple oncogenic mutations with relevance to targeted therapy in non-small-cell lung cancer. J Mol Diagn. 13: 74-84.

  4. Dias-Santagata D, Akhavanfard S, David SS, et al. (2010). Rapid targeted mutational analysis of human tumours: a clinical platform to guide personalized cancer medicine. EMBO Mol Med. 2: 146-58.