Primers in FOXG1 used in this study
| Primer | Primer sequence (5′ to 3′) | PCR product size (bp) | Primer concentration (µM) | Annealing temperature/MgCl2 concentration/betaine (°C/mM/M) |
| Sequencing | ||||
| Exon 1-1F | AGC CTG CGG TCC AAC TGC GC | 565 | 0.8 | 58/1.5/2 |
| Exon 1-1R | CAG CCC GTC CGC TTT AGC CC | |||
| Exon 1-2F | GCC GCC GCA GCA GCA GCA GC | 657 | 58/1.5/2 | |
| Exon 1-2R | CGC AGC TTG CCC GTG GTG CC | |||
| Exon 1-3F | ACG CTC AAC GGC ATC TAC GA | 621 | 65/1.5/0 | |
| Exon 1-3R | AGA GCA GGG CAC CGA CAG GC | |||
| Exon 1-4F | TCA GAA CTC GCT GGG CAA CA | 641 | 65/1.5/0 | |
| Exon 1-4R | TCT GAC CAG CTA GAT CCC AC | |||
| Exon 2F | AAG CCT GTT GGA GAA TTA GA | 390 | 61/1.5/0 | |
| Exon 2R | TCT CTA GCA CCT TTT AAC TC | |||
| Exon 3F | ATA ACC CAC CTC AGA TTT TG | 439 | 61/1.5/0 | |
| Exon 3R | GCA TCC TTT CAG CTA CGT CT | |||
| Exon 4F | TTT ATT GAA GTC TGG AGG AG | 587 | 63/1.5/0 | |
| Exon 4R | GTC AAC ACT GTC CAC TAC CC | |||
| Exon 5F | GGT GAA CTT AAC AAC ATA GC | 690 | 61/2.5/0 | |
| Exon 5R | TTA GCC AGG ATG GTC TCG AT | |||
| QMPSF | ||||
| Exon 1 -B-F | CG GCA AGT ACG AGA AGC CGC | 179 | 0.6 | 58/2/2 |
| Exon 1 -B-R | AT GGA GTT CTG CCA GCC CTG C | |||
| Exon 2 -F | TT CAC CAG ATA ACC TGC CCC | 230 | 0.15 | |
| Exon 2 -R | AC GTG TGT GCC CAA AAG AGA G | |||
| Exon 4 -F | GG GCC TTG GGA GAT GGA A | 196 | 0.3 | |
| Exon 4 -R | CC TCT TTT CAA CCT TCT GCA AAA C | |||
| Exon 5 -F | TC AGT GTG CTG TTG TCA ATC AGA | 0.4 | ||
| Exon 5 -R | AC CAA GAA TTC AGA CAT AGG AGA GA | |||
| Control BRCA1 exon 20-R | GC TCC ACT TCC ATT GAA GGA AG | 250 | 0.3 | |
| Control BRCA1 exon 20-F | AG CGG CCC ATC TCT GCA AAG |
For each amplicon in the QMPSF, forward (F) and reverse (R) primers are equimolar. The size of the fluorescent PCR products for the QMPSF includes the sequences added on the 5′ side: 5′-CGTTAGATAG-3′ (forward primers) and 5′-GATAGGGTTAT-3′ (reverse primers). For the QMPSF multiplex PCR, forward primers are 5′ labelled with the fluorescein 6-FAM dye. We amplified short fragments of the multi-exon coding region of FOXG1 corresponding to exon 1, exon 3 (exons 2 and 3 are very close at the genomic level), exon 4 and exon 5. A standard amplicon (exon 20 of BRCA1) was included in the multiplex PCR for analysis of fluorescent profiles.
For sequencing of exon 4, internal primers (F: 5′-TAGAACGATAGGGCCT-3′; R: 5′-AAACTCTCCCTCTGCTC-3′) were used instead of M13F/M13R because of a polyT tract at the end of intron 3.
QMPSF, quantitative multiplex PCR of short fluorescent fragments.