Table 3.
Primers combinationsfor CYP2A6 genotyping real-time PCR genotyping (Method 2.1 and 2.2)
| Assay | Primer Name | Locationa | Direction | Sequence |
|---|---|---|---|---|
| First amplifications
| ||||
| 5′ region template: | 2A65Pr1F | 5′ flanking (−1417) | F | ACC TAG ACT TAA TCT TCC CGT ATA C |
| CYP2A6*9, *31, *24, *20, *23 | 2A6in5R | Intron 5 (3638) | R | GGC CTG TGT CAT CTG CCT |
| Modified 5′ region: | 2Ain1F-L | Intron 1 (316) | F | GAT CTT GGG ATG TCC AGC TCC |
| CYP2A6*12, *34 | 2A6in5R | Intron 5 (3638) | R | GGC CTG TGT CAT CTG CCT |
| 3′ region template: | 2A6in6F1 | Intron 6 (4892) | F | ATTTCCTGCTCTGAGACC |
| CYP2A6*17, *35, *1B | 2A6R13 | 3′ flanking (8548) | R | GCC TCC CAT AGT GCT ATA ATT AAC A |
|
| ||||
| Second amplifications
| ||||
| CYP2A6*9 | 2A6*9wtR-Sybr-Mb | 5′ flanking (−29) | R | GCT GGG GTG GTT TGC CTC TA |
| 2A6*9vR-Sybr-Mb | 5′ flanking (−29) | R | GCT GGG GTG GTT TGC CTC TC | |
| 125M13BEV-B | 5′ flanking (−146) | F | CCC AAG CTA GGC AGG ATT CAT G | |
|
| ||||
| CYP2A6*31 | 2A6*31wtR-Sybr | Exon 1 (35) | R | AGC AAG GCC ACC AGA AGC AT |
| 2A6*31vR-Sybr | Exon 1 (35) | R | AGC AAG GCC ACC AGA AGC AG | |
| 2A61F-L | Exon 1 (−155) | F | TGG CTG TGT CCC AAG CTA GGC A | |
|
| ||||
| CYP2A6*24 | 2A6*24wtR-Sybr-Mb | ExIn 2 (619) | R | ACC CCC TCA CCA TAG CCT TTG AAT AC |
| 2A6*24vR-Sybr-Mb | ExIn 2 (619) | R | ACC CCC TCA CCA TAG CCT TTG AAT AG | |
| 2A6ex2-505F | Exon 2 (505) | F | TGC TGT GTG GAC ATG ATG CCG TCA G | |
|
| ||||
| CYP2A6*20 | 2A6*20wtR-sybr-Mb | Exon 4 (2160) | R | CAA CAG TGA CAG GAA CTC TTT GTA CT |
| 2A6*20vtR-sybr-Mb | Exon 4 (2160) | R | CAA CAG TGA CAG GAA CTC TGT ACT | |
| 2A6in3F-L | Intron 3 (1967) | F | CCC TGC CTC CTG GAA TTC TGA C | |
|
| ||||
| CYP2A6*23 | 2A6ex42161Fw-Mb | Exon 4 (2139) | F | CAA AGA GTT CCT GTC ACT GTC GC |
| 2A6ex42161Fv-M-Lb | Exon 4 (2137) | F | GA CAA AGA GTT CCT GTC ACT GTC GT | |
| 5M13FOR-H2 | Intron 4 (2260) | R | GCA GTT GGC AGG TTG TGG TAG G | |
|
| ||||
| CYP2A6*12 | 2A6in1/ex2-L | In1/Ex2 (434) | F | CCACCTCCATCAGATCAGTGAGC |
| 2A7in1/ex2-L | In1/Ex2 (952) | F | TCGCCTCCATCAGTTCAGTGAGT | |
| 2A6R-667 | Intron 2 (667) | R | GAACACTGAGACCTTCGTGTCCA | |
|
| ||||
| CYP2A6*34 | 2A6-2572F | Intron 4 (2572) | F | TCAACCGGCCTCCTGCATA |
| 2A7-2978F | Intron 4 (2978) | F | AAC CCG CCT CCT GCA TG | |
| 2A6-2862R | Intron 4 (2862) | R | GTTTACCTATCCAAGTGGGATGCACT | |
|
| ||||
| CYP2A6*17 | 2A6*17Fwt-Sybr-Mb | Exon 7 (5042) | F | ACG AGA TCC AAA GAT TTG GAG CCG |
| 2A6*17Fv-Sybr-Mb | Exon 7 (5042) | F | ACG AGA TCC AAA GAT TTG GAG CCA | |
| 2A6in7-17R2 | Intron 7 (5198) | R | GGA TGC TGG GGA CAC AGA GAG | |
|
| ||||
| CYP2A6*35 | 2A6*35wtR-Sybr | Exon 9 (6479) | R | CCA GGC CTT CTC CGA AAC AGT T |
| 2A6*35vR-Sybr | Exon 9 (6479) | R | CCA GGC CTT CTC CGA AAC AGT A | |
| 2A6in8-6395F | Intron 8 (6395) | F | CGA GGC TGC ACT GAG AGT GG | |
|
| ||||
| CYP2A6*1B | 2A6*1Bwt | 3′ flanking (6719) | F | ACT GGG GGC AGG ATG GC |
| 2A6*1Bvar-L | 3′ flanking (7101) | F | GGG TAT AAG AAT GGG GGG AAG ATG CG | |
| 2A6R6944 | 3′ flanking (6944) | R | GTG GCA ATT AGG TGA GCG TGC AAT G | |
a
CYP2A6 primer position is based on CYP2A6 +1ATG, and CYP2A7 primer position is based on CYP2A7 +1ATG
b
Mismatch primer at third position from 3′end, underlined in sequence