Fig. 1

Validation of the established pyrosequencing analysis method using a novel dispensation order on cell lines and FFPE tissues. DNA from six tumor cell lines and four FFPE tissues was analyzed in parallel by pyrosequencing and dideoxy sequencing. a Examples of pyrograms obtained on the NSCLC cell lines A549 (i and ii), H1650 (iii), and H1975 (iiii) for mutational analysis of EGFR exon 19 or exon 21. b Examples of pyrograms obtained on the CRC cell lines HT-29 (i), SW480 (iii), and DLD-1 (iiii) as well as the NSCLC cell line A549 (ii) for mutational analysis of KRAS exon 2, codons 12 and 13. c To test the suitability of pyrosequencing for clinical samples, the assay was used to analyze EGFR or KRAS mutations in the following known mutated FFPE tissues: NSCLC M1 (i), NSCLC M2 (ii), CRC M1 (iii), and CRC M2 (iiii). All of the targeted mutations occurring in the cell lines and FFPE tumor tissues could easily be distinguished by pyrosequencing. The results for the six cell lines and four FFPE tissues were 100 % concordant between the established pyrosequencing and dideoxy sequencing methods. The target nucleotide sequences are labeled and underlined. The horizontal axis of each pyrogram, from left to right, indicates the order of reagent addition. E represents enzyme. S represents substrate. Mutation points are indicated by light shading. The vertical axis represents the luminescence intensity, of which the peak heights are proportional to the number of each nucleotide incorporated at one time. Sites of variation are indicated by arrows. Data are representative of five independent analyses of the same sample