PCR Variations for Unknown Sequence Variants

Most of the techniques discussed above are used to screen for sequence variants (both mutations and polymorphisms) based on previous knowledge of the variant; i.e., the sequence of the variant is either known or defined by the experimental conditions. By contrast, there is an evolving interest in both research and clinical molecular pathology to identify sequence variants by scanning without prior knowledge of their existence; i.e., the sequence of the variant is unknown. Sequencing is the ultimate screening technique, but is costly and labor-intensive. The goal of the scanning techniques described below (denaturing gradient gel electrophoresis [DDGE], and temperature gradient gel electrophoresis [TGGE], heteroduplex analysis [HA], single-strand conformation polymorphism [SSCP], denaturing high-performance liquid chromatography [DHPLC], and protein truncation test [PTT]) is to identify specimens with possible variant sequences, thereby reducing costs relative to sequencing. Should an unknown variant be detected, for example by a shift in the mobility of the PCR product on a gel or capillary, the PCR product with altered mobility is isolated and sequenced. Melting-temperature analysis in real-time PCR also can be used to identify unknown sequence variants.

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