Efficient IDUA Gene Mutation Detection with Combined Use of dHPLC and Dried Blood Samples

Table 1

PCR amplification conditions.

Exon 9	Amplicon size (bp)	GC content (%)	Primers (5^'→ 3^')	PCR program
First PCR	379	78	F-GGAGCGAGTGGTGGGAGG	97°C/5 min
			R-GACACTCAGGCCTCGGCTC	96 ° C/1 min	35X
				60 ° C/1 min
Nested PCR	208	77	F-GGCGGCTGGGCAACGACC	72 ° C/1 min
			R-GTGGGCGCGGGTGTCGTC	72°C/10 min

PCR reactions were performed using PCR MasterMix (Promega) without any additives, 20 pmol of forward primer and 20 pmol of reverse primer; in the first PCR, 4 𝜇L (4-5 ng) of DNA was used in a 10 𝜇L final volume; in the nested PCR, 1 𝜇L of a 1/10 dilution of the first PCR product was used in a 20 𝜇L final volume.