Supporting Information_ Methods S1
Direct bidirectional sequencing of the complete coding regions and
exon-intron boundaries of genes SHH (MIM 600725), SIX3 (MIM 603714),
ZIC2 (MIM 603073), and TGIF (MIM 602630), were performed using the Big
Dye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City,
CA), and the automated sequencer ABI Prism 3130XL (Applied Biosystems).
Primers sets and PCR protocols for amplification of SHH, SIX3, and TGIF
genes were used as described elsewhere (El-Jaick et al., 2007a; El-Jaick et
al., 2007b). Primers sets and PCR protocols for amplification of ZIC2 gene
were also described elsewhere (Costa-Lima, 2008; Savastano, 2009),
however are also described here to increase availability. For amplification of
ZIC2 gene, a total of four primer pairs were used: Z1 (foward) 5’ATCGGGAGCGGGAGTCGAG-3’ and Z2 (reverse) 5’AGCACATTCTGCGAGCCGTG- 3’ for amplification of the first part of exon 1;
Z3.1 (forward) 5’-AACTCCACCCGGGACTTCCTGTTC-3’ and Z4.1 (reverse)
5’-CCACAGCCTGAGTCTCAG-3’ for amplification of second part of exon 1;
Z5 (forward) 5’-TGCAGCCAGCGCCGATGTTTGC-3’ and Z6 (reverse) 5’TGAGGTGGTCCGGGCCAGTGC-3’ for amplification of exon 2; Z7 (forward)
5’-AGCTGCACTCACACCCAGTC-3’ and Z8 (reverse) 5’AAGGTGCCCTCGCTGCTAGCTG-3’ for amplification of coding region of
exon 3. Reactions were performed in a PTC-220 thermocycler (MJ Research,
Waltham, MA) in a final volume of 35 μL using 60–100 ng of genomic DNA,
200 μM dNTP, 20 pmol of each primer, 1× PCR buffer (Invitrogen, Carlsbad,
CA), 0.5× enhancer (Invitrogen, Carlsbad, CA), 1.5 mM MgSO4 (Invitrogen,
Carlsbad, CA), and 2.5 U of AmpliTaq Applied Biosystems). For amplification
of ZIC2 exon 3, 3x enhancer (Invitrogen, Carlsbad, CA) was used. PCR
cycling parameters were 95°C for 5 minutes, followed by 95°C for 45 sec,
62°C for 1 min, 72°C for 45 sec, for 35 cycles, with a final step of 72°C for 7
minutes. Annealing step for amplification of exon 3 was performed at 55˚C.
Full COLD-PCR (Li et al., 2008) of ZIC2 exon 3 was performed to confirm the
mutation in the father. Reagents concentration was the same as in normal
PCR, and cycling conditions were 95°C for 5 minutes, 25 cycles of (95°C for
45 sec, 55°C for 1 min, 72°C for 45 sec), then 30 cycles of (95°C for 45 sec,
70°C for 8 min, 80°C for 3 sec, 55°C for 1 min, 72°C for 45 sec), and a final
step of 72°C for 7 minutes.
Sequences were aligned with reference coding sequences (Genbank
cDNA accession numbers: SHH: NM_000193.2; SIX3: NM_005413.2; ZIC2:
NM_007129.2, TGIF: NM_007129.3) using Sequencher (Gene Codes
Corporation Ann Arbor), and mutation was confirmed on a new PCR and
sequencing reaction.
References for Supporting Information_ Methods S1
Costa-Lima MA. 2008. Estudo Molecular do Gene ZIC2 em Pacientes com
Defeito de Fechamento do Tubo Neural e Holoprosencefalia. [Tese de
Doutorado]. Rio de Janeiro: Universidade Federal do Rio de Janeiro.
161 p.
El-Jaick KB, Fonseca RF, Moreira MA, et al. 2007a. Single median maxillary
central incisor: new data and mutation review. Birth Defects Res A Clin
Mol Teratol 79(8):573-580.
El-Jaick KB, Powers SE, Bartholin L, et al. 2007b. Functional analysis of
mutations in TGIF associated with holoprosencephaly. Mol Genet
Metab 90(1):97-111.
Li J, Wang L, Mamon H, Kulke MH, Berbeco R, Makrigiorgos GM. 2008.
Replacing PCR with COLD-PCR enriches variant DNA sequences and
redefines the sensitivity of genetic testing. Nat Med 14:579-584.
Savastano CP. 2009. Estudos Moleculares em Holoprosencefalia
[Dissertação de Mestrado]. Rio de Janeiro: Universidade Federal do
Rio de Janeiro. 96 p.