title: Single-stranded conformation polymorphism (SSCP)-driven indirect sequencing in detection of short deletion.
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.
authors.affiliation: Department of Medicine, Jagiellonian University School of Medicine, 8 Skawinska Str., 31-066, Cracow, Poland.
authors.forename: Michal
authors.initials: M
authors.lastname: Natkaniec
authors.forename: Daniel P
authors.initials: DP
authors.lastname: Potaczek
authors.forename: Marek
authors.initials: M
authors.lastname: Sanak
mesh_terms: D001483:Base Sequence; D005787:Gene Frequency; D008722:Methods; D011110:Polymorphism, Genetic; D020641:Polymorphism, Single Nucleotide; D018807:Polymorphism, Single-Stranded Conformational; D017422:Sequence Analysis, DNA; D017384:Sequence Deletion / Q000235:genetics*
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