Table 1

Primers in FOXG1 used in this study

PrimerPrimer sequence (5′ to 3′)PCR product size (bp)Primer concentration (µM)Annealing temperature/MgCl2 concentration/betaine (°C/mM/M)
Sequencing
 Exon 1-1FAGC CTG CGG TCC AAC TGC GC5650.858/1.5/2
 Exon 1-1RCAG CCC GTC CGC TTT AGC CC
 Exon 1-2FGCC GCC GCA GCA GCA GCA GC65758/1.5/2
 Exon 1-2RCGC AGC TTG CCC GTG GTG CC
 Exon 1-3FACG CTC AAC GGC ATC TAC GA62165/1.5/0
 Exon 1-3RAGA GCA GGG CAC CGA CAG GC
 Exon 1-4FTCA GAA CTC GCT GGG CAA CA64165/1.5/0
 Exon 1-4RTCT GAC CAG CTA GAT CCC AC
 Exon 2FAAG CCT GTT GGA GAA TTA GA39061/1.5/0
 Exon 2RTCT CTA GCA CCT TTT AAC TC
 Exon 3FATA ACC CAC CTC AGA TTT TG43961/1.5/0
 Exon 3RGCA TCC TTT CAG CTA CGT CT
 Exon 4FTTT ATT GAA GTC TGG AGG AG58763/1.5/0
 Exon 4RGTC AAC ACT GTC CAC TAC CC
 Exon 5FGGT GAA CTT AAC AAC ATA GC69061/2.5/0
 Exon 5RTTA GCC AGG ATG GTC TCG AT
QMPSF
 Exon 1 -B-FCG GCA AGT ACG AGA AGC CGC1790.658/2/2
 Exon 1 -B-RAT GGA GTT CTG CCA GCC CTG C
 Exon 2 -FTT CAC CAG ATA ACC TGC CCC2300.15
 Exon 2 -RAC GTG TGT GCC CAA AAG AGA G
 Exon 4 -FGG GCC TTG GGA GAT GGA A1960.3
 Exon 4 -RCC TCT TTT CAA CCT TCT GCA AAA C
 Exon 5 -FTC AGT GTG CTG TTG TCA ATC AGA0.4
 Exon 5 -RAC CAA GAA TTC AGA CAT AGG AGA GA
 Control BRCA1 exon 20-RGC TCC ACT TCC ATT GAA GGA AG2500.3
 Control BRCA1 exon 20-FAG 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.