Single-stranded conformation polymorphism (SSCP)-driven indirect sequencing in detection of short deletion |
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Authors: | Michal Natkaniec Daniel P Potaczek Marek Sanak |
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Institution: | (1) Department of Medicine, Jagiellonian University School of Medicine, 8 Skawinska Str., 31-066 Cracow, Poland |
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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. |
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Keywords: | FCER1A Deletion Mutational screening SSCP |
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