Fumagalli et al. BMC Cancer 2010, 10:101 http://www.biomedcentral.com/1471-2407/10/101 Page 3 of 14 Foster City, CA) yielded the best DNA based on the quality, quantity, and the performance on the mass spectrometer (data not shown). The extraction was per- formed as recommended by the manufacturer, with two exceptions; the protease digestion was extended over- night and the elution volume was increased to 150 ul to maximize the total amount of DNA recovered. Addi- tional protease was added to samples incompletely digested after the overnight treatment. DNA was mea- sured with fluorescence, using the Quant-iT ™ Pico- Green® dsDNA Assay Kit (Invitrogen, Carlsbad, CA) and the InfiniteF200 fluorometer (Tecan, Mannedorf, Switzerland). As a positive control for known mutations and to test the performance of the platform, annotated cell line DNAs (A2058, HS578T, HL60, MCF7, MDAMB231, NCI-H1299, NCI-H1395, UACC-893) were purchased from American Type Culture Collection (ATCC, Mana- ssas, VA, US). Two cell lines, SKBR3 and MCF-7, were grown in culture and cell pellets were fixed in formalin, andembedded in paraffin and DNAs were isolated as described for the clinical samples. Mass Spec Type Plex Technology and the OncoCarta Panel For mutation detection, the Sequenom platform and the OncoCarta mutation panel were used and the pro- tocol provided by Sequenom (San Diego, CA) was fol- lowed with minor modifications. A schematic of the procedure is shown in Fig. 1. A Tecan Evo liquid handler was used to normalize the DNA samples and to set up the PCR reactions. The amount of DNA Figure 1 Methodology for mutation detection. Genomic DNA from the samples is amplified by PCR, resulting in copies of both mutant and wildtype alleles. Shrimp Alkaline Phosphatase removed excess nucleotides from the sample wells. Primer extension was performed using terminator nucleotides A, C, T, G, each with distinct masses. This linear amplification results in sequences proportional to the alleles that can be distinguished by mass spec (Maldi-Tof Separation).