High recovery of cell-free methylated DNA based on a rapid bisulfite-treatment protocol

BMC Molecular Biology

Table 1 Primer and beacon sequences

Primer/beacon	Sequence
MLH1 UF	TGT GAI AAA AAA TGT GAA GGG
MLH1 DF	GAA GAT ATT AGA TTT TAT GGG TTA TTT
MLH1 R	CAA CTI AAT TTT AAC AAA ATA ATC T
MLH1 UDF	ACC AGA TTT TAT GGG TCA TCC
MLH1 UDR	TTC TAT TAA CGT ACG GAC G
MLH1 beacon	(FAM)CGC GAA TGT GGA AGG AAA AGT GAG TGT CGC(Dabcyl)
RASSF1A MF	GGG AGG CGT TGA AGT C
RASSF1A MR	CCC GTA CTT CGC TAA CTT TAA ACG
RASSF1A M beacon	(HEX)CGC GAT TCG + TT + C G + GT TCG CTC GCG(Dabcyl)
RASSF1A UMF	TTT TGT ATT TAG GTT TTT ATT GTG T
RASSF1A UMR	CCC ATA CTT CAC TAA CTT TAA ACA
RASSF1A UM beacon	(FAM)CGC GAG + TT + TG + GTT + TG + TG + TTTC GCG(Dabcyl)
MEST MF	TGT CGC GGT AAT TAG TAT ATT TC
MEST MR	AAC CCG CGC AAA ACG ACG
MEST M beacon	(HEX)CGC GAT TAC + GAA AC + G CAA CTA CCG ATC GCG(Dabcyl)
MEST UMF	GTG TTG TTG TGG TAA TTA GTA TAT TTT
MEST UMR	AAC CCA CAC AAA ACA ACA CCA
MEST UM beacon	(FAM)CGC GAG + TA + G T + TG + TG + T TT + T GTT CGC G(Dabcyl)

MLH1 UF and MLH1 R concurrently amplify deaminated and undeaminated MLH1 promoter. MLH1 DF and MLH1 R amplify deaminated MLH1. MLH1 UDF and MLH1 UDR amplify undeaminated MLH1 promoter. A common molecular beacon (MLH1 beacon) was used for detection of the 3 products. RASSF1A MF and RASSF1A MR amplify methylated RASSF1A. RASSF1A UMF and RASSF1A UMR amplify unmethylated RASSF1A. Methylated and unmethylated products were detected by RASSF1A M beacon and RASSF1A UM beacon, respectively. MEST MF and MEST MR amplify methylated MEST, whereas MEST UMF and MEST UMR amplify unmethylated MEST. Methylated and unmethylated products were detected by MEST M beacon and MEST UM beacon, respectively. To ensure high specificity the probes designed to discriminate between methylated and unmethylated products contain LNA nucleotides at positions marked with +.

ISSN: 1471-2199