Table 1 Primer sets for determination of perforin CpG methylation by pyrosequencing
From: Determination of quantitative and site-specific DNA methylation of perforinby pyrosequencing
Amplicon* (bp) | PCR primers† (5' to 3') | Sequencing primers‡ | CpG site # and nucleotide position∞ |
|---|---|---|---|
A (236) | FW: TTG ATT TTA TAG GTG AGG AAA TTA RV: TTC CAA CTA TCA CCC ATA ACC TAA | TTT TAT AGG TGA GGA AAT TA A Y GTTTAGAAAGGGGGTTGATATTTAT Y GT Y GTGAGGTATA Y GG ATT TTT ATG TTT TTT AAA T Y GGTTTTTTTGTTA | 1, -1365; 2, -1339; 3, -1336; 4, -1325 6, -1231 |
B (226) | FW: ATG TTG AGG TTG TGA GGA GTT TT RV: AAA TTC CAA AAT CCT CTC TTT AAT | GAA GTT TTG TAA AGT ATT TG Y GGGAAAAGAG | 5, -1267 |
C (196) | FW: TTG GAA AGT GAT TAG GAG GTT GTA RV: CAA ACT CCA AAC CAC ATA TAA CAT | AGA GGG TGG GGA TATT G Y GGAGAGAAGATGGGGTTAGATTT Y G GTT TTT GTT TTT GTA AGA GT AGGGA Y GGAAGTAGGGATATAAA Y G | 7, -1110; 8, -1088 9, -1045; 10, -1027 |
D (91) | FW: GAG GTT TTT ATG GGT GGA GTG AT RV:CAC CTC CTC CCT TAC CCA ACT A | TTG GGG GGT AAA ATT ATA Y GGTTTT T | 11, -876 |
E (256) | FW: TTT TGA GTG GGA GAA GAG AGA TGT RV:CCC CAC CCT AAC CTC AAA CA | GTG AGA GTG GTT TGG TAG TAT Y GGAGG TTG GTT TTA GTT TTG TTG A GGT Y GTGGGT AGG ATA GTT AGT GGT TTT TA Y GTTGGTTTTAGTTTTGTTG | 12, -776 13, -774 14, -720 |
F (167) | FW: TGG AGG TTA TTG GTT GTT TTT ATA RV: TAA CCA TTC CCT CCT CCC TAA ATA | GGT TAT TGG TTG TTT TTA TA AAG Y GAGGAG T AGGAGTTTTTGTT Y GAGGAATATGTTTGGAGTT Y GG | 15, -691; 16, -670; 17, -650 |
G (300) | FW: TTA GGG AGG AGG GAA TGG TTA TAG RV: AAC CAA CAA AAC CAT CTC CTT ACT | TAG TTT ATA TTG TTG GTG TA TAAT Y GAGTTGTTTAAGTTT Y GG Y GGTTTGG Y G | 18, -397; 19, -381; 20, -378; 21, -370 |
H (300) | FW: TTT AGG GAG GAG GGA ATG GTT ATA RV: ACC AAC AAA ACC ATC TCC TTA CTT | TAC TTC TAA TAC ACA ACA TC R CATATATAAAATATAAAAAACAAACAAAAAC R AC R AC | 24, -313; 23, -345; 22, -348 |
I (233) | FW: GGG GAT TTA GGG TAT ATA GG RV: AAA CCC TAC CAA TCC ACA CTA CT | GGG GAT TTA GGG TAT AT AGG Y GGAGGAGGG Y GGGG Y GTTGAGGATTTTGAGATT Y GGT | 28, -219; 27, -229; 26, -234; 25, -253 |
J (237) | FW: TGG TTT TGT TGG TTT GTT TAT TAA RV: CCC CAA CTA TAA TCA CAA ATC CTT | CAA AAC CAA AAA CTC ATC T ACC R AATAAAACTACTAAAACTC R CCT CAA CCC TCA TCC R ACTCCCCACTAACAACCCTCAAAAAAC R AAC | 30, -180; 29, -200 32, -122; 31, -150 |
K (97) | FW: TTG AGG ATA GGG TGG GTG TT RV: CCA CCA CTC ACA TCA CTT CTA CTT | GGA TAG GGT GGG TGT T Y GTGGGAGGGGAGAGTATAAAGGATTTGTGATTATAGTTGGGGG Y | 33, -92; 34, -48 |