Use of a competitive probe in assay design for genotyping of the UGT1A1 *28 microsatellite polymorphism by the smart amplification process.
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.
Watanabe
Jun
J
RIKEN, Yokohama, Japan. nabe-jun@gsc.riken.jp
Mitani
Yasumasa
Y
Kawai
Yuki
Y
Kikuchi
Takeshi
T
Kogo
Yasushi
Y
Oguchi-Katayama
Atsuko
A
Kanamori
Hajime
H
Usui
Kengo
K
Itoh
Masayoshi
M
Cizdziel
Paul E
PE
Lezhava
Alexander
A
Tatsumi
Kenji
K
Ichikawa
Yasushi
Y
Togo
Shinji
S
Shimada
Hiroshi
H
Hayashizaki
Yoshihide
Y
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
D015342:DNA Probes; D014453:Glucuronosyltransferase; D000099148:UGT1A1 Enzyme