Table 1

Primers and PCR annealing temperature for SMAD amplifications

SMAD	Exon/region	Sense primer (5′–3′)	Antisense primer (5′–3′)	Size (bp)	Ta
1	67–901	tgctgactgggttactttttta	aagcaggtgggggcgtatca	835	55°C
	162–543	caaatctcttctgctgtcct	ttccagtggttttagttcat	382	53°C
	497–870	gtcatttactgccgtgtgtg	gaaagggcttcctgggtctg	374	55°C
	799–1714	ctctcccaatagcagttacc	gctcctttgtcagttctcaa	916	54°C
	838–1249	cagcagcacctaccctcact	cctcccctccaacataataa	412	55°C
	1229–1704	attggaaaaggagttcatct	cagttctcaatcgtgtctga	476	52°C
2	1 part 1	tccgctccctccgtcttccatac	cgcgcgcccgcagccctacc	340	67°C
	1 part 2	gacggcggccgggagtgttt	gcgggcgcccaggctttacc	168	63°C
	2	ggtgaaggaagtattctgta	aatgctatgccttattttac	183	50°C
	3	ttttacatcatggtattttg	attttacattaaggaaacat	282	47°C
	4	aatttagcccatttgactgc	gctattccaagaaacagata	473	48°C
	5	ttggatttcttgaacttttt	aacttgaatgcttatgaaca	225	49°C
	6	gctgtgcttgatttgtttta	atgcgtctcaacttctctaa	214	47°C
	7	tttttaaatccttttgtttt	ttatttggctattcattagg	199	46°C
	8	aatctatttttggcttgaat	aatgcctacattatgagtat	342	47°C
	9	ctcatttgtattttgtttca	gttgacatgataggtttatg	198	48°C
	10	atattctaaaacttgtaacc	agaatgcaatgaaacataat	290	47°C
	11	ctgcctgtggacttgaat	tcttgaacttttggatag	150	48°C
3	1	gtcgtccatcctgcctttca	tggtgatgcacttggtgttg	137	55°C
	2–3 part 1	atggccggttgcaggtgtcc	aggcaggccaggcagcatac	205	59°C
	2–3 part 2	cccccggacagttctacctc	tgccgcccacgtgcctacct	183	58°C
	4	gaccaccttccttctgattc	atgaccctgcatgactgacc	120	55°C
	5	tgtctcacctcgcaggttct	tgcacaaggagatactcacc	80	55°C
	6	gtagcccaccctctgtccac	agccacccataccgatgtg	250	58°C
	7	gaggcgtgcggctctactac	tgcctgtgcggctcgtttac	155	58°C
	8	gccctgtttctgtgtttttg	aggcagcacccataactgac	206	55°C
	9	cccacccctttccctatt	aagacacactggaacagc	150	58°C
5	1 part 1	tctccgaagatttgtgtcaa	ctaaagatctgggaatagtg	237	50°C
	1 part 2	aggacagccgagtaaatgtg	tttttccaaattcttctcag	260	50°C
	2	gacttttgatttttgttttt	tgggagctgaaatggacttc	274	51°C
	3	agattttaattattattttt	atgaagtgagtanttctctt	173	45°C
	4	tctgtgtctggtttgttcac	attaaatgtaggaaaatgac	272	47°C
	5	ttttaaggtgttcatctgta	tgttagaggtcacaactcac	284	50°C
	6	aagagggatttgtgatgata	ttaaaacaagtccactaaca	227	49°C

Cycle conditions: four minutes 94°C initial denaturation; 94°C for 30 seconds, 0.5–1 minute at specified temperature (Ta), 72°C for 0.5–1 minute for 35 cycles; and a final extension step of seven minutes at 72°C. For SMAD1, the gene was amplified in two segments initially (67–901, 799–1714, GenBank U59912), followed by two nested PCRs within each of those regions as shown.