Use of a competitive probe in assay design for genotyping of the UGT1A1 *28 microsatellite polymorphism by the smart amplification process |
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Authors: | Watanabe Jun Mitani Yasumasa Kawai Yuki Kikuchi Takeshi Kogo Yasushi Oguchi-Katayama Atsuko Kanamori Hajime Usui Kengo Itoh Masayoshi Cizdziel Paul E Lezhava Alexander Tatsumi Kenji Ichikawa Yasushi Togo Shinji Shimada Hiroshi Hayashizaki Yoshihide |
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Affiliation: | RIKEN, Yokohama, Japan. nabe-jun@gsc.riken.jp |
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Abstract: | A key feature of the smart amplification process version 2 (SMAP-2) is the ability to suppress mismatch amplification by using a unique asymmetric primer design and Thermus aquaticus MutS (Taq MutS). However we report here that use of SMAP-2 for polymorphism determination of the UGT1A1 *28 allele required a further ancillary approach for complete background suppression. The UGT1A1 *28 allele is a microsatellite copy number polymorphism. This is the first reported SMAP-2 assay designed for genotyping genetic variations of microsatellites. We found that by the addition of a primer to the amplification reaction, called a competitive probe (CP), assay specificity could be significantly enhanced. Including sample preparation time and use of a CP-enhanced SMAP-2 assay, we could rapidly detect the UGT1A1 *28 polymorphism within 60 min. To test our method, we compared results from PCR sequencing and the CP-enhanced SMAP-2 assay on 116 human blood samples for UGT1A1 *28 polymorphism and demonstrated perfect concordance. These results illustrate the versatility of SMAP-2 for molecular diagnostics and provide a new approach for enhancing SMAP-2 assay specificity. |
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