title: Use of a competitive probe in assay design for genotyping of the UGT1A1 *28 microsatellite polymorphism by the smart amplification process.
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.
authors.affiliation: RIKEN, Yokohama, Japan. nabe-jun@gsc.riken.jp
authors.forename: Jun
authors.initials: J
authors.lastname: Watanabe
authors.forename: Yasumasa
authors.initials: Y
authors.lastname: Mitani
authors.forename: Yuki
authors.initials: Y
authors.lastname: Kawai
authors.forename: Takeshi
authors.initials: T
authors.lastname: Kikuchi
authors.forename: Yasushi
authors.initials: Y
authors.lastname: Kogo
authors.forename: Atsuko
authors.initials: A
authors.lastname: Oguchi-Katayama
authors.forename: Hajime
authors.initials: H
authors.lastname: Kanamori
authors.forename: Kengo
authors.initials: K
authors.lastname: Usui
authors.forename: Masayoshi
authors.initials: M
authors.lastname: Itoh
authors.forename: Paul E
authors.initials: PE
authors.lastname: Cizdziel
authors.forename: Alexander
authors.initials: A
authors.lastname: Lezhava
authors.forename: Kenji
authors.initials: K
authors.lastname: Tatsumi
authors.forename: Yasushi
authors.initials: Y
authors.lastname: Ichikawa
authors.forename: Shinji
authors.initials: S
authors.lastname: Togo
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Shimada
authors.forename: Yoshihide
authors.initials: Y
authors.lastname: Hayashizaki
mesh_terms: D001681:Biological Assay / Q000379:methods*; D015342:DNA Probes / Q000235:genetics*; D005838:Genotype; D014453:Glucuronosyltransferase / Q000235:genetics*; D018895:Microsatellite Repeats / Q000235:genetics*; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D020641:Polymorphism, Single Nucleotide / Q000235:genetics*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA / Q000379:methods*; D000099148:UGT1A1 Enzyme
chemical_list: D015342:DNA Probes; D014453:Glucuronosyltransferase; D000099148:UGT1A1 Enzyme