Abstract
To seek for novel rare and/or sporadic mutations within genomic neighborhood of common −344 C>T (rs2427827) FCER1A proximal promoter polymorphism, which by its prevalence could have masked the presence of less frequent genetic variants in our previous single-stranded conformational polymorphism (SSCP) mutational search study, SSCP screening was repeated using primers fixing −344 C>T variant. The genomic region surrounding −344 C>T polymorphism was confirmed to be fairly conservative and only a single sporadic mutation (present in 1 out of 196 chromosomes), a 6-bp deletion −262 to 257 del CTAGAC, was found. From the methodological point of view, we demonstrated a successful detection of a short-length polymorphism using SSCP screening in a population, in which the same genomic region contained frequent single-nucleotide polymorphic variant. In conjunction with subsequent cloning of aberrantly migrating PCR products and SSCP-driven indirect sequencing of the clones, this method offers a superior (to direct sequencing of PCR products) detection of rare mutations. The cost-effective method applied by us enables a reliable characterization of infrequent (thus present only in heterozygotic form) short-length variance of the sequence which are difficult to interpret by direct sequencing.
Similar content being viewed by others
References
Zhang L, Chen H, Lan X, Zhang C, Zhang L, Zhang A, Zhang Q, Lei C, Zhang H (2008) The novel 5 bp deletion polymorphism in the promoter region of bovine ACRP30 gene. Mol Biol Rep. doi:10.1007/s11033-008-9260-1
Potaczek DP, Nishiyama C, Sanak M, Szczeklik A, Okumura K, Ogawa H (2008) FCER1A gene exon 1A polymorphisms in Japanese and Polish subjects—a comparative analysis of haplotypes. Allergy 63:626–627. doi:10.1111/j.1398-9995.2008.01675.x
Potaczek DP, Sanak M, Mastalerz L, Milewski M, Gawlewicz-Mroczka A, Szczeklik A (2007) Genetic polymorphisms of the novel FCER1A gene region: relation to total serum IgE levels. Ann Allergy Asthma Immunol 98:500–501
Shikanai T, Silverman ES, Morse BW, Lilly CM, Inoue H, Drazen JM (2002) Sequence variants in the FcεRI alpha chain gene. J Appl Physiol 93:37–41
Potaczek DP, Sanak M, Mastalerz L, Setkowicz M, Kaczor M, Nizankowska E et al (2006) The α-chain of high-affinity receptor for IgE (FcεRIα) gene polymorphisms and serum IgE levels. Allergy 61:1230–1233. doi:10.1111/j.1398-9995.2006.01195.x
Kim SH, Ye YM, Lee SK, Park HS (2006) Genetic mechanism of aspirin-induced urticaria/angioedema. Curr Opin Allergy Clin Immunol 6:266–270
Bae JS, Kim SH, Ye YM, Yoon HJ, Suh CH, Nahm DH et al (2007) Significant association of FcεRIα promoter polymorphisms with aspirin-intolerant chronic urticaria. J Allergy Clin Immunol 119:449–456. doi:10.1016/j.jaci.2006.10.006
Sanak M, Potaczek DP, Mastalerz L, Nizankowska E, Szczeklik A (2007) FCERIA gene promoter polymorphisms: lack of association with aspirin hypersensitivity in whites. J Allergy Clin Immunol 119:1280–1281. doi:10.1016/j.jaci.2007.01.030
Kanada S, Nakano N, Potaczek DP, Maeda K, Shimokawa N, Niwa Y et al (2008) Two different transcription factors discriminate the −315C>T-polymorphism of the FcεRI α-chain gene: binding of Sp1 to −315C and of a high mobility group-related molecule to −315T. J Immunol 180:8204–8210
Acknowledgments
D. P. P. was awarded Foundation for Polish Science (FNP) START stipend.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Natkaniec, M., Potaczek, D.P. & Sanak, M. Single-stranded conformation polymorphism (SSCP)-driven indirect sequencing in detection of short deletion. Mol Biol Rep 36, 1545–1547 (2009). https://doi.org/10.1007/s11033-008-9347-8
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s11033-008-9347-8