Table 1.

Primers for amplifying and sequencing PITX2 and FOXC1.

Gene	Exon	Forward primer (5′-3′)	Reverse primer (5′-3′)	Product size (bp)	Annealing temperature (°C)
PITX2	2	GGCCGCCGCTTCTTACA	CACTGGCGATTTGGTTCTGATTT	254	63
	3	CTGCCTCGCCCCTCCCCCTCCTC	TTCAAGCAGCAGGCGCGTCAGGTC	350	64
	4	GGGGCAGTAGCCAAGGACT	CAGCTAAGCGGGAATGTCTG	289	64
	5	GGCGCGGACACCCACACTTG	GCGCTGCCTTCCACATTCTCT	487	65
	6-A	GCGCTGCCTTCCACATTCTCT	GGTGGATAGGGAGGCGGATGTAAG	346	63
	6-B	GGTGGATAGGGAGGCGGATGTAAG	CGACGGGCTACTCAGGTTGTTCA	256	64
	6-C	TCCCGGGCTCCAGTCTCAACAG	TTTCTTTAGTGCCCACGACCTTCT	355	64
	SSCP-1	CGACGACATGTACCCAGGCTATTC	GCGACGGGCTACTCAGGTTGTT	257	68
	SSCP-2	GGCGCGGACACCCACACTTG	CTGCCGCCTTTGCCGCTTCTTCTT	269	68
	SSCP-3	CTGGCCCTGGTATCTTGGTGTGC	GGTGGATAGGGAGGCGGATGTAAG	346	63
FOXC1	1-A	CCCGGACTCGGACTCGGC	AAGCGGTCCATGATGAACTGG	429	63
	1-B	GCGCACGCCGAGCAGTA	CACCGCGTCCTTCTTCTTGA	390	60
	1-C	CACCCTGAACGGCATCTACCA	AGGCTGCTGCTGCTGCTGTCG	504	64
	1-D	GCCCGTGCGCATCCAGGACATCAA	GCTGCCCGCGCTGGAGGTCTGG	463	64
	1-E	AGGGCTTCAGCGTGGACAACATCA	GGTGGGCCGCAGGGTGGTG	482	65
	1-F	CAAGCCATGAGCCTGTACG	GGGTTCGATTTAGTTCGGCT	502	60

Primer sequences, sizes of PCR products, and annealing temperatures used for the amplification are listed. Primers 2–6 were used to amplify and sequence the PITX2-coding segments. The sequencing of exon 6 of PITX2 was performed with three overlapping primers A–C. Primers SSCP-1 to SSCP-3 were used to amplify the novel variations detected in PITX2 for heteroduplex-SSCP analysis. The single FOXC1 exon was sequenced with six overlapping primers, A–F.