Table 1 Primer sequences and cycling details for multiplex PCR and SNaPshot

Multiplex PCR - Primers and Cycling Conditions

EGFR Ex 18 FWD

5′- CCCCCCCAGCTTGTGG -3′

95^OC - 3 min

45 cycles of:

94^OC - 30 s

63^OC - 90 s

72^OC - 90 s

Final extension

72^OC - 10 min

4 ^OC - end

EGFR Ex 18 REV

5′- ACCGTGCCGAACGCAC -3′

EGFR Ex 20 FWD

5′- CTCTCCCTCCCTCCAGGAAG -3′

EGFR Ex 20 REV

5′- TTCCCGGACATAGTCCAGGA -3′

EGFR Ex 21 FWD

5′- CGCAGCATGTCAAGATCACAG -3′

EGFR Ex 21 REV

5′- TGGCTGACCTAAAGCCACCT -3′

SNaPShot Assay - Primers and Cycling Conditions

EGFR-2155R

5′- TGGTTAGATGGAACGCACCGGAGC -3′

96^OC - 10 s

25 cycles of:

50^OC 5 s

60^OC - 30 s

Final:

4^OC - ∞ min

EGFR-2156R

5′- TAGATGTGGTTAGATGCGAACGCACCGGAG -3′

EGFR-2369R

5′- TGGTTAGATGTGGTTAGATGGAAGGGCATGAGCTGC -3′

EGFR-2573 F

5′- ATGTGGTTAGATGTGGTTAGATGAAGATCACAGATTTTGGGC -3′

EGFR-2582 F

5′- GATGTGGTTAGATGTGGTTAGATGTGGTTAGATGTGGGCTGGCCAAAC -3′

Sizing Assay - Primers and Cycling conditions

EGFR Ex19 FWD

5′ - GCACCATCTCACAATTGCCAGTTA-3

98^OC - 3 min

45 cycles of:

98^OC - 30 s

60^OC - 60 s

72^OC - 60 s

Final extention:

72^OC 10 min

4^OC - ∞ min

EGFR Ex19 REV

5′-/6FAM/AAAAGGTGGGCCTGAGGTTCA-3)