PCR primer sequences for mutation analyses in the GCDH gene
| Primer name | Sequence (5′-3′) | Sequencing | DGGE Primer 5′ tail | ||||||
|---|---|---|---|---|---|---|---|---|---|
| Primer 5′ tail | Product size (bp) | ||||||||
| A DGGE analysis and sequencing of all exons | |||||||||
| Exon 1 | GCDH-1F | TCGTTGCTCCGCTCGCTCTG | P51 | 216 | — | ||||
| GCDH-1R | AGTCCCTAAACCCCCAGTTC | M13 | 65mer | ||||||
| Exon 2 | GCDH-2F | AGTGTGGGGTCGGGAGTGTG | P51 | 260 | 40mer | ||||
| GCDH-2R | CGGGCAGCTCTCGGATTCTG | P172 | — | ||||||
| Exon 3 | GCDH-3F | GAAGGGAGGGCACAGTGAT | P51 | 280 | 60mer | ||||
| GCDH-3R | GCGGAGGAGCAGTCTCAG | P172 | — | ||||||
| Exon 4 | GCDH-4F | ATAGCCACCCCACCTCAAG | P51 | 261 | — | ||||
| GCDH-4R | AAGGAGGAAGAGGCTTTCAGA | P172 | 40mer | ||||||
| Exon 5 | GCDH-5F | TGTCCTTATTCAGCCCTGTC | M13 | 338 | 40mer | ||||
| GCDH-5R | GACTGTCTTCCTTCCACCAG | P172 | — | ||||||
| Exon 6 | GCDH-6F | GGCAGCCTTGTGACTTTGTC | P172 | 272 | — | ||||
| GCDH-6R | AGTCGGTGAGGGGTCTGAC | M13 | 40mer | ||||||
| Exon 7 | GCDH-7F | TGGGCAGGTGGTGAACAG | P172 | 373 | — | ||||
| GCDH-7R | CCGCATCCGCAGGTGAC | P51 | 40mer | ||||||
| Exon 8 | GCDH-8F | CTTTCCCTGCTTCAGAGTTG | P172 | 264 | 40mer | ||||
| GCDH-8R | CCACACCCCCAGAGAATC | M13 | — | ||||||
| Exon 9 | GCDH-9F-a | GACGGGGTGGGAGAGTG | P172 | 359 | / | ||||
| GCDH-9R-a | AGCCCATCAAGGACAAGAG | P51 | / | ||||||
| Exon 9 | GCDH-9F-b | GCCTCCCCTCGCTCTTAC | / | 40mer | |||||
| GCDH-9R-b | CTCCAGGAAGACACAAGGTC | / | — | ||||||
| Exon 10 | GCDH-10F | GCCCACTGGTCCCTCATTG | P172 | 378 | 40mer | ||||
| GCDH-10R | TACCCCCTCCCCAGACACT | M13 | — | ||||||
| GCDH-c.1173-R | TACTCGTCAGAAATCCCAGT | / | / | ||||||
| Exon 11 | GCDH-11F | AAAACTCCAAACCGACTCTGT | M13 | 418 | / | ||||
| GCDH-11R-a | GAAGCTGCTATTTCAGGGTAA | P172 | / | ||||||
| Exon 11 | GCDH-11F | AAAACTCCAAACCGACTCTGT | / | 40mer | |||||
| GCDH-11R-b | CGCCACCTCCCTTTCTAAG | / | — | ||||||
| GCDH-c.1482-F | TCCCTTCTGAAGTCGATC | / | / | ||||||
| B Primer 5′ tails for sequencing | |||||||||
| M13 | TATGTAAAACGACGGCCAGT | ||||||||
| P172 | TATTATAGGGCGAATTGGGT | ||||||||
| P51 | TATAAGGGAACAAAAGCTGG | ||||||||
| C Primer 5′ tails for DGGE | |||||||||
| 40mer | CGCCCGCCGCGCCCCGCGCCCGTCCCGCCGCCCCCGCCCG | ||||||||
| 60mer | CGCCCGCCGCCCCGCCGCCCGCTCGCCGCCCGCCGCGCCCCTGCCCGCCGCCCCCGCCCG | ||||||||
| 65mer | CGCCCCGCCCGCCGCCCCGCCGCCCGCTCGCCGCCCGCCGCGCCCCTGCCCGCCGCCCCCGCCCG | ||||||||
The appropriate universal sequence (B) or GC clamp (C) was attached 5′ to the specific primers (A). The ACRS primersGCDH-c.1173-R (together with 10F, product size 253 bp) and GCDH-c.1482-F (together with 11R-a, product size 141 bp) were used for the analysis of polymorphisms G391G and c.1482A>G, respectively. Primers for sequencing and DGGE of exons 9 and 11 were at different positions.