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
RIKEN, Yokohama, Japan.
Biotechniques. 2007 Oct;43(4):479-84. doi: 10.2144/000112563.
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.
智能扩增过程版本2（SMAP-2）的一个关键特性是能够通过使用独特的不对称引物设计和嗜热水生栖热菌MutS（Taq MutS）来抑制错配扩增。然而，我们在此报告，使用SMAP-2进行UGT1A1 *28等位基因的多态性测定需要进一步的辅助方法来完全抑制背景。UGT1A1 *28等位基因是一种微卫星拷贝数多态性。这是首次报道的用于微卫星基因分型的SMAP-2检测方法。我们发现，通过在扩增反应中添加一种引物，称为竞争探针（CP），可以显著提高检测的特异性。包括样品制备时间和使用CP增强的SMAP-2检测方法，我们能够在60分钟内快速检测UGT1A1 *28多态性。为了测试我们的方法，我们比较了116份人类血液样本的PCR测序结果和CP增强的SMAP-2检测方法对UGT1A1 *28多态性的检测结果，并证明了完全一致性。这些结果说明了SMAP-2在分子诊断中的多功能性，并为提高SMAP-2检测特异性提供了一种新方法。
