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Fig. 2 | Journal of Experimental & Clinical Cancer Research

Fig. 2

From: The mutation rates of EGFR in non-small cell lung cancer and KRAS in colorectal cancer of Chinese patients as detected by pyrosequencing using a novel dispensation order

Fig. 2

Limit of detection and assay linearity. Mixtures of DNA from EGFR or KRAS cell lines were analyzed in triplicate and in parallel by pyrosequencing and dideoxy sequencing methods to analyze the limit of detection and assay linearity (Additional file 1: Figures S2–S5, Tables S5–S6). a To assess the limit of detection, cell line DNA was mixed to produce samples containing differing proportions of mutant alleles. The established pyrosequencing method allows for 2 % of EGFR exon 19 c.2235_2249del15 mutant alleles (i), 3 % of EGFR exon21 c2573 T>G mutant alleles (ii), 2 % of KRAS exon2 c.35G>T mutant alleles (iii), and 5 % of KRAS exon 2 c.38G>A mutant alleles (iiii) to be detected with confidence. The percentages on the horizontal axis indicate the calculated percentages of mutant alleles present, while the vertical axis indicates the percentage of nucleotide. The white column represents the percentage of detected wild-type nucleotides; the red column within the white column represents the percentage composition of the detected mutant nucleotides. Arrows indicate the DNA dilutions in which mutations could be reliably detected above background noise. WT means wild-type. b To assess the assay linearity of pyrosequencing, theoretical peak heights, calculated from the initial mutant allele peak percentage in undiluted mutant cells, were correlated with actual peak heights generated for each dilution. For mutational analysis of EGFR exon 19 c.2235_2249del15 (i), KRAS exon 2 c35 G>T (iii), and c.38 G>A (iiii), Pearson’s correlation (r) = 0.99; for mutational analysis of EGFR exon 21 c.2573 T>G (ii), r = 0.96, indicating a linear relationship. A, adenine; G, guanine; T, thymine; C, cytosine. Data are from three independent experiments

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