Consequences of primer binding-sites polymorphisms on genotyping practice

Estefânia M. Martins, Laura Vilarinho, Sofia Esteves, Mónica Lopes-Marques, António Amorim, Luísa Azevedo

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DOI: 10.4236/ojgen.2011.12004                

Abstract

Herein we investigated the effect of primer binding site polymorphisms in achieving correct genotyping when a mismatch occurs in distinct positions of the primer sequence. For that purpose primer sequences were designed in order to carry either allelic form at the 3’ end and at 3 bp, 5 bp and 7 bp apart from the 3’ end of an intronic polymorphism (rs2247836) observed in phenylalanine hydroxylase (PAH) gene. For one of the alleles annealing failure was obtained when the mismatch occurs at all the four primer-site locations. Primer sequences carrying the alternative SNP allele resulted to be less specific as the distance to the primer-3’ end was increased. Altogether, these results revealthat effects in the extension of the annealing failure is allele and mismatch-position dependent.

Keywords

Primer Binding-site; SNP; PAH Gene; Molecular Genetics

Conflicts of Interest

The authors declare no conflicts of interest.

References

[1]	Koboldt, D.C., Miller, R.D. and Kwok, P.Y. (2006) Distribution of human SNPs and its effect on high- throughput genotyping. Human Mutation, 27, 249-254. doi:10.1002/humu.20286
[2]	Quinlan, A.R. and Marth, G.T. (2007) Primer-site SNPs mask mutations. Nature Methods, 4, 192. doi:10.1038/nmeth0307-192
[3]	Alves, C., et al., (2001) VWA STR genotyping: Further inconsistencies between Perkin-Elmer and Promega kits. International Journal of Legal Medicine, 115, 97-99. doi:10.1007/s004140100215
[4]	Wong, L.J., et al. (2001) Novel SNP at the common primer site of exon IIIa of FGFR2 gene causes error in molecular diagnosis of craniosynostosis syndrome. American Journal of Medical Genetics, 102, 282-285. doi:10.1002/ajmg.1461
[5]	Jeffrey, G.P., et al. (1999) Polymorphism in intron 4 of HFE may cause overestimation of C282Y homozygote prevalence in haemochromatosis. Nature Genetics, 22, 325-326. doi:10.1038/11892
[6]	Frazer, K.A., et al. (2007) A second generation human haplotype map of over 3.1 million SNPs. Nature, 449, 851-861. doi:10.1038/nature06258