Table 2.

Amplification and Restriction Endonuclease Digestion to Confirm Mutations/SNPs^[Note]

						Digestion Fragmentsa(bp)
Mutation/SNP	Exon	Primers	AmpliconSize(bp)	Enzyme	No. ofControlDNAs	Genotype 11	Genotype 12	Genotype 22
c.1402C→T (p.R468W)	13	52 and 53	336	BtsI	95	CC (95): 283, 53	CT (0): 336, 283, 53	TT (0): 336
c.1579_1581del (p.L527del)	13	52 and 53	336	BseRI	86	Wt (86): 223, 113	Het (0): 336, 223, 113	Mut (0): 336
c.228delC	4	32 and 33	451	StuI	94	Wt (94): 359, 92	Het (0): 451, 359, 92	Mut (0): 451
c.413C→T (p.S138F)	5	32 and 33	451	BmgBI	95	CC (95): 352, 99	CT (0): 451, 352, 99	TT (0): 451
c.1538T→A (p.V513E)	13	52 and 53	336	AluI	48	TT (29): 196, 140	TA (19): 336, 196, 140	AA (0): 336

Note.— The region of SLC34A3 flanking the mutation/SNP was amplified by PCR in a final volume of 20 μl by use of the indicated primers (table A1) (Qiagen PCR kit); the thermal cycler conditions after an initial denaturation at 94°C for 5 min were 35 cycles at 94°C for 1 min, 60°C or 65°C for 1 min, and 72°C for 3 min, followed by a final extension at 72°C for 10 min. PCR products were then subjected to restriction endonuclease digestion to obtain the indicated genotype-specific fragments on 2% agarose/TAE gel electrophoresis.

^aGenotypes are followed by the respective fragment(s), given in bp, and the number of controls with a particular genotype is in parentheses. Het = heterozygote; Mut = mutant; Wt = wild type.