Detection of three closely located single nucleotide polymorphisms in the EAAT2 promoter: comparison of single-strand conformational polymorphism (SSCP), pyrosequencing and Sanger sequencing

Table 1 Pyrosequencing primers and conditions used in the study

Oligonucleotide	Sequence 5′-3′	Product (bp)	Annealing T (°C)	Annealing T (°C)
EAAT2F	GGGGCTAAACCTTGCAATCC	180	65	None
EAAT2R	CTGCCACCTGTGCTTTGC	180	65	None
EAAT2PyroF-BIO	GGGGCTAAACCTTGCAATC	166	60	5′ Biotin
EAAT2PyroR	GAGTGGCGGGAGCAGAGA	166	60	None
EAAT2PyroSeq1	GGGTGTGTGCGCGCC	N/A		None
EAAT2PyroSeq2	CCGCACACGCGCACG	N/A		None
Target sequence for pyrosequencing (1)		T/GGGGGAGGCGGTGGAGGCCG/TCTG
Nucleotide dispensation order (1)		CGTGCAGCGTGAGCGTGC
Target sequence for pyrosequencing (2)		G/ATGTGTGCGCGCC
Nucleotide dispensation order (2)		CAGTGTGT

Primer pair EAAT2PyroF-BIO/EAAT2PyroR were used to generate biotinylated PCR products flanking SNPs g.-200C > A; g.-181A > C and g.-168C > T. Primers EAAT2PyroSeq1 (to detect g.-200C > A;-181A > C) and EAAT2PyroSeq2 (to detect g.-168C > T) were used for pyrosequencing. In the dispensation order the nucleotides used as negative controls are underlined. The nucleotide change in the Target sequence for pyrosequencing is indicated in bold.

ISSN: 2730-6844