Supplementary section 1
PCR and Sequencing conditions
PCR reactions were run in 25 l reactions with 0.3 l of each primer using the Qiagen
Multiplex PCR kit (Qiagen). Samples were subjected to initial denaturation at 95C
for 15 min, 38 cycles at 94C for 45 s, annealing at 60C for 90 s, and elongation at
72C for 90 s, followed by a final elongation step for 10 min at 72C. The PCR
product was enzymatically treated with ExoSAP-IT (USB, Cleveland, OH, USA).
Forward sequence primer was identical to PCR primer, reverse sequencing primer
were different (see supplementary table 2),all samples were sequenced in both
directions utilizing the BigDye Terminator Sequencing Kit, version 1.1. The sequence
reactions were analyzed on an ABI Prism 3100 genetic analyzer with Sequencing
Analysis software, version 3.7.
Primer sequences for the amplification and sequencing of KRAS exon 2
Gene
and
exon
Forward primer
Reverse Primer
KRAS GTGTGACATGTTCTAATATAGTCA GAATGGTCCTGCCCAGTAA
exon
2
GTCCTGCACCAGTAATATGC
KRAS
exon
2 seq
Size of
PCR
Annealing
temperature
product (°C)
(bp)
218
60
Primer sequences for the amplification and sequencing of PIK3CA exon 9 and
Gene
and
exon
Forward primer
Reverse Primer
PIK3CA
Exon 9
GGGAAAAATATGACAAAGAAAGC
CTGAGATCAGCCAAATTCAGTT
PIK3CA
Exon 9
seq
PIK3CA
Exon 20
PIK3CA
Exon 20
seq
GGAAAAATATGACAAAGAAAGCTTATAAG
ACAGAGAATCTCCATTTTAGCAC
GCTCCAAACTGACCAAACTGTTC
TGGAATCCAGAGTGAGCTTTC
CTCAATGATGCTTGGCTCTG
CCTGCTGAGAGTTATTAACAGTGC
Size of
PCR
Annealing
product
(bp)
279
(°C)
349
temperature
60
60