Phenotypic Variation in a Four-Generation Family with Aniridia Carrying a Novel PAX6 Mutation

Table 1

PCR primer sequences.

Exon	Forward primer	Chromosomal coordinates	Reverse primer	Chromosomal coordinates	Fragment size
4	TTCCAGTACTTTGTTTCAAGCCCC	chr11:31,806,650-31,806,673	AAACTCGGGCGGGCTGTTCTTAAG	chr11:31,806,160-31,806,183	514 bp
5	CCTCTTCACTCTGCTCTCTT	chr11:31,802,874-31,802,893	ATGAAGAGAGGGCGTTGAGA	chr11:31,802,637-31,802,656	258 bp
5a	TGAAAGTATCATCATATTTGTAG	chr11:31,801,999-31,802,021	GGGAAGTGGACAGAAAACCA	chr11:31,801,785-31,801,804	237 bp
6	TGAAAGTATCATCATATTTGTAG	chr11:31,801,999-31,802,021	AGGAGAGAGCATTGGGCTTA	chr11:31,801,507-31,801,526	515 bp
7	CAGGAGACACTACCATTTGG	chr11:31,800,880-31,800,899	CAGGCCTTCAAATGCAGTCTCACC	chr11:31,800,557-31,800,580	343 bp
8	GGGAATGTTTTGGTGAGGCT	chr11:31,794,891-31,794,910	CAAAGGGCCCTGGCTAAATT	chr11:31,794,540-31,794,559	371 bp
9	GTAGTTCTGGCACAATATGG	chr11:31,794,136-31,794,155	GTACTCTGTACAAGCACCTC	chr11:31,793,950-31,793,969	206 bp
10	GTAGACACAGTGCTAACCTG	chr11:31,793,820-31,793,839	CCCGGAGCAAACAGGTTTAA	chr11:31,793,597-31,793,616	243 bp
11	TTAAACCTGTTTGCTCCGGG	chr11:31,793,597-31,793,616	AGTGCGAAAAGCTCTCAAGGGTGC	chr11:31,793,326-31,793,349	291 bp
12	GCTGTGTGATGTGTTCCTCA	chr11:31,790,891-31,790,910	TTTCCCTTTTCAATCCCCATCCCC	chr11:31,790,566-31,790,589	345 bp
13	CATGTCTGTTTCTCAAAGGGA	chr11:31,790,055-31,790,075	CCCCAGTGGTACAATACAGGACAC	chr11:31,789,782-31,789,805	294 bp

UCSC human genome, hg38 accessed 9/10/2017. In a 25 μL PCR reaction for each coding exon, 0.3 μL of AmpliTaq Gold (Applied Biosystems), with 2 μL of 25 mM MgCl2, 0.2 of 25 mM dinucleotide triphosphate, and 0.5 μL of 25 mM primers were used. For exon 4, we used 5 μL of Q-solution (Qiagen) and changed MgCl2 volume to to 2.5 μL and accounted for this from the water to keep the reaction volume at 25 μL. In all of the amplicons, genomic DNA was denatured at 94°C for 10 minutes then amplified by 36 cycles of 94°C denaturation for 30 seconds, 60°C annealing for 30 seconds, and 72°C extension for 1 minute with final extension of 72°C for 10 minutes.