Open Access Methodology article

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

Shavanthi Rajatileka1, Karen Luyt24, Maggie Williams3, David Harding4, David Odd25, Elek Molnár6 and Anikó Váradi1*

Author Affiliations

1 Centre for Research in Biosciences, Department of Biological, Biomedical and Analytical Sciences, Faculty of Health and Applied Sciences, University of the West of England, Bristol BS16 1QY, UK

2 Neonatal Neuroscience, School of Clinical Sciences, University of Bristol, St Michael’s Hospital, Southwell Street, Bristol BS2 8EG, UK

3 Bristol Genetics Laboratory, Pathology Sciences, Blood Sciences and Bristol Genetics, Southmead Hospital, Bristol BS10 5NB, UK

4 Regional Neonatal Intensive Care Unit, St Michael’s Hospital, University Hospitals NHS Trust, Bristol BS2 8EG, UK

5 Neonatal Intensive Care Unit, Southmead Hospital, North Bristol NHS Trust, Bristol BS10 5NB, UK

6 School of Physiology and Pharmacology University of Bristol, Medical Sciences Building, University Walk, Bristol BS8 1TD, UK

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BMC Genetics 2014, 15:80  doi:10.1186/1471-2156-15-80

Published: 5 July 2014



Single-strand conformational polymorphism (SSCP) is still a frequently used genotyping method across different fields for the detection of single nucleotide polymorphisms (SNPs) due to its simplicity, requirement for basic equipment accessible in most laboratories and low cost. This technique was previously used to detect rs4354668:A > C (g.-181A > C) SNP in the promoter of astroglial glutamate transporter (EAAT2) and the same approach was initially used here to investigate this promoter region in a cohort of newborns.


Unexpectedly, four distinct DNA migration patterns were identified by SSCP. Sanger sequencing revealed two additional SNPs: g.-200C > A and g.-168C > T giving a rise to a total of ten EAAT2 promoter variants. SSCP failed to distinguish these variants reliably and thus pyrosequencing assays were developed. g.-168C > T was found in heterozygous form in one infant only with minor allele frequency (MAF) of 0.0023. In contrast, g.-200C > A and -181A > C were more common (with MAF of 0.46 and 0.49, respectively) and showed string evidence of linkage disequilibrium (LD). In a systematic comparison, 16% of samples were miss-classified by SSCP with 25-31% errors in the identification of the wild-type and homozygote mutant genotypes compared to pyrosequencing or Sanger sequencing. In contrast, SSCP and pyrosequencing of an unrelated single SNP (rs1835740:C > T), showed 94% concordance.


Our data suggest that SSCP cannot always detect reliably several closely located SNPs. Furthermore, caution is needed in the interpretation of the association studies linking only one of the co-inherited SNPs in the EAAT2 promoter to human diseases.

EAAT2 promoter; Single nucleotide polymorphism; Genotyping; Pyrosequencing; SSCP; Premature newborns; Dried blood spots; Glutamate regulation