mutation detection. (A) Target DNA from two single tumor cells (SC1, SC2) successfully pre-amplified by PCR with the expected bands for PIK3CA exon 9 (216 bp) and exon 20 (269 bp). (B) Second round of amplification using the pre-amplified PCR products from (A) as new DNA templates to separately amplify exon 9 (using original primers, 216 bp) and exon 20 (using internal primers, 192 bp). P1 = PCR product from first round of amplification; P2 = PCR product from second round of amplification. (C) Sanger sequencing results for PIK3CA mutation G1633A on exon 9: two MCF7 single cells shown here carry the G1633A heterozygous mutation; Normal DNA, BT474 single cells, and single WBCs are wild type (G/G); PIK3CA G1633A mutations were detected in single DTCs and CTCs from breast cancer patient 12. The G1633A mutation was distinguishable in the chromatogram from the adjacent A1634C peak from a known pseudogene on chromosome 22 that may be co-amplified with these primers , as in sample MCF7.2. (D) Sanger sequencing results for PIK3CA mutation A3140G on exon 20: BT20 cells show the mutation but MCF7 cells, BT474 cells, and CTCs are wildtype for this mutation hotspot.