Table 3.

Pyrosequencing primers and conditions used in the study

Oligonucleotide	Sequence 5′-3′	Product size (bp)	T (°C)	Modifications
rs1835740PyroF	CTCATTCGTTTTCTGCCTGTTG	300	60	None
rs1835740PyroR-BIO	TCTTGCATATTTGAGCAGACTTTG			5′Biotin
rs1835740PyroSeq	CACAACTTGATTCCAATCT	N/A		None
Target sequence	GC/TGTATGTAGATT
Nucleotide dispensation order	AGCTCGTAT
rs4354668PyroF-BIO	GGGGCTAAACCTTGCAATC	166	60	5′Biotin
rs4354668PyroR	GAGTGGCGGGAGCAGAGA			None
rs4354668PyroSeq	GGGTGTGTGCGCGCC	N/A		None
Target sequence	T/GGGGGAGGCGGTGGAGGCC
Nucleotide dispensation order	CGTGCAGCGTGAGCGTGC

Primer pair rs1835740PyroF/rs1835740PyroR-BIO and rs4354668PyroF-BIO/rs4354668PyroR were used to generate biotinylated PCR products flanking SNPs rs1835740 and rs4354668, respectively. Primers rs1835740PyroSeq and rs4354668PyroSeq were used for pyrosequencing. The target sequence and the order of nucleotide dispensation for each pyrosequencing assay are listed. In the dispensation order the nucleotides used as negative controls for pyrosequencing are underlined. In optimal pyrosequencing conditions these nucleotides are not incorporated into the target DNA sequence and thus their addition do not generate peak on the pyrogram (see also Figure 2). The nucleotide change in the target sequence is indicated in bold.