Competitive amplification of differentially melting amplicons (CADMA) improves KRAS hotspot mutation testing in colorectal cancer

Table 1 Details of the CADMA assays

Mutation	Cell line	Primers (introduced mutations are underlined)	Primer concentrations	Annealing temperature
c.34 G > A	A549	Mutation specific forward:	400 nM	57°C
		GAATATAAACTTATGGTAGTTGGAGATA
		Overlapping forward:	100 nM
		ATGACTGAATATAAACTTGTGGTAGTTG
		Common reverse:	400 nM
		ACTGTCAAGGCACTCTTGCCTAC
c.34 G > T	NCI-H23	Mutation specific forward:	400 nM	60°C
		GAATATAAACTTGTAGTAATTGGAGCTT
		Overlapping forward:	150 nM
		ATGACTGAATATAAACTTGTGGTAGTTG
		Common reverse:	400 nM
		ACTGTCAAGGCACTCTTGCCTAC
c.34 G > C	PSN1	Mutation specific forward:	400 nM	60°C
		GAATATAAACTTGTAGTAATTGGAGCTC
		Overlapping forward:	100 nM
		ATGACTGAATATAAACTTGTGGTAGTTG
		Common reverse:	400 nM
		ACTGTCAAGGCACTCTTGCCTAC
c.35 G > A	LS174T	Mutation specific forward:	400 nM	60°C
		GAATATAAACTTGTGGTAATTGGAGATGA
		Overlapping forward:	150 nM
		ATGACTGAATATAAACTTGTGGTAGTTG
		Common reverse:	400 nM
		ACTGTCAAGGCACTCTTGCCTAC
c.35 G > T	SW480	Mutation specific forward:	400 nM	64°C
		GAATATAAACTTGTAGTAATTGGAGCTGT
		Overlapping forward:	150 nM
		ATGACTGAATATAAACTTGTGGTAGTTG
		Common reverse:	400 nM
		ACTGTCAAGGCACTCTTGCCTAC
c.35 G > C	RPMI8226	Mutation specific forward:	400 nM	58°C
		GAATATAAACTTGTAGTAATTGGAGCTGC
		Overlapping forward:	100 nM
		ATGACTGAATATAAACTTGTGGTAGTTG
		Common reverse:	400 nM
		ACTGTCAAGGCACTCTTGCCTAC
c.38 G > A	DLD-1	Mutation specific forward:	400 nM	62°C
		AAACTTGTGGTAGTTGGAGATGGTTA
		Overlapping forward:	150 nM
		ATGACTGAATATAAACTTGTGGTAGTTG
		Common reverse:	400 nM
		ACTGTCAAGGCACTCTTGCCTAC

ISSN: 1471-2407