Microarray-based method for detecting methylation changes of p16Ink4a gene 5’-CpG islands in gastric carcinomas

Table 1 Nucleotide sequences of methylated and unmethylated probes analyzed in oligonucleotide microarray

p16 CpG sites		Oligonucleotide sequences	Tm (°C)
#4-6	M:	5’-NH2-(T)10-CAACCGCCGAACGCAC-3’	56
	U:	5’-NH2-(T)10-CAACCACCAAACACAC-3’	50
#8-10	M:	5’-NH2-(T)10-CAACCGCCGAACGCAC-3’	61
	U:	5’-NH2-(T)10-CCACCACCCACTACCTA-3’	53
#11-13	M:	5’-NH2-(T)10-CCGCCGCCGACTCCAT-3’	61
	U:	5’-NH2-(T)10-CCACCACCAACTCCAT-3’	53
#15-17	M:	5’-NH2-(T)10-AACCGCGACCGTAACCAA-3’	58
	U:	5’-NH2-(T)10-AACCACAACCATAACCAA-3’	50
#18,19	M:	5’-NH2-(T)10-TCTACCCGACCCCGAACC-3’	60
	U:	5’-NH2-(T)10-TCTACCCAACCCCAAACC-3’	55
#20,21	M:	5’-NH2-(T)10-AACAACGCCCGCACCTC-3’	57
	U:	5’-NH2-(T)10-AACAACACCCACACCTC-3’	50
#22,23	M:	5’-NH2-(T)10-ACAACGCCCCCGCCTC-3’	59
	U:	5’-NH2-(T)10-ACAACACCCCCACCTC-3’	52
#24,25	M:	5’-NH2-(T)10-AACTATTCGATACGTTAAAC-3’	48
	U:	5’-NH2-(T)10-AACTATTCAATACATTAAAC-3’	39
#26-28	M:	5’-NH2-(T)10-ATCGACCTCCGACCGTAAC-3’	55
	U:	5’-NH2-(T)10-ATCAACCTCCAACCATAAC-3’	49

Table 2 MSP primers used for amplification of p16 gene CpG island. MS and US represent methylated and unmethylated sense primers, respectively. MA1 and MA2, UA1 and UA2 represent methylated and unmethylated antisense primers, respectively

Primer sets	Sequences (5’→3’)	Size bp	Annealing temp (°C)
MS	TTATTAGAGGGTGGGGCGGATCGC	234	65
MA1	CCACCTAAATCGACCTCCGACCG
US	TTATTAGAGGGTGGGGTGGATTGT	234	60
UA1	CCACCTAAATCAACCTCCAACCA
MS	TTATTAGAGGGTGGGGCGGATCGC	150	65
MA2	GACCCCGAACCGCGACCGTAA
US	TTATTAGAGGGTGGGGTGGATTGT	151	60
UA2	CAACCCCAAACCACAACCATAA