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 +.

Pedersen et al. BMC Molecular Biology 2012 13:12   doi:10.1186/1471-2199-13-12

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