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.

Introduction

Genetic variations that influence pharmacokinetic and pharmacodynamic properties of drugs are now being introduced into clinical decision making for treatment of certain human cancers (1). Tailored chemotherapy based on patient genetics may have significant potential to improve prognosis for many cancers. Supporting this point, and to minimize the possibility of severe adverse drug reactions, the U.S. Food and Drug Administration (FDA) has required the drug industry to publicly provide single nucleotide polymorphism (SNP) data examined in the process of procuring a drug license.

A key requirement for the development of individualized medicine is the ability to rapidly and conveniently test patients for gene polymorphisms and/or mutations. Previously, we developed a rapid polymorphism detection system called the smart amplification process version 2 (SMAP-2) (2). To suppress any misamplification of highly related target species (e.g., a wild-type sequence versus a SNP variant), SMAP-2 utilizes two fundamental technologies. The first is a unique asymmetric design of the primers flanking the target sequence that are engaged in later stage self-priming events; these two primers are referred to as the folding primer (FP) and turn-back primer (TP). The second approach to minimize background amplification uses Thermus aquaticus MutS (Taq MutS) in combination with an isothermal amplification procedure. The Taq MutS protein binds to mismatched nucleotides in erroneously primed dsDNA and prevents further amplification by inhibiting the dissociation of the mismatched DNA duplex by the strand-displacing DNA polymerase. These fundamental design features are the reason the SMAP-2 assay, unlike any other method, give a reliable diagnostic result based exclusively on amplification alone.

Previously we engineered SMAP-2 assays and reported the detection of various SNPs and mutations, such as ALDH2(G1543A), CYP2C19* 2(G681A), DIO2(T274C), ADRB3 (C192T), EGFR exon21(T2573G), EGFR exon18(G2155T), and EGFR exon19(deletion A to G) (2,3). Each of these genetic variations were either SNPs, singlenucleotide mutations, or microdeletions. However, microsatellite polymorphisms that are typically copy number differences in 2–4 nucleotide repeats are a very important class of genetic variations found in many genes. One example of a micro-satellite polymorphism that has been well studied and linked to a pharmacokinetic phenotypic outcome is the UGT1A1*28 allele. The TATA box in the promoter of this allele generally includes a wild-type sequence of (TA)₆TAA. However, the UGT1A1*28 allele has a two-base pair insertion (TA) resulting in the sequence (TA)₇TAA and is associated with impaired pharmacokinetics of the drug irinotecan and its metabolites (4). We found it difficult to detect this microsatellite polymorphism accurately using SMAP-2, because it was not possible to suppress exponential background amplification of the wild-type allele completely using the conventional primer design approach of SMAP-2.

In previous reports, the assay specificity of allele-specific PCR, oligonucleotide microarrays, and other techniques were shown to be enhanced with the use of a competitive probe (CP) (5,6,7). It was reported that addition of a CP dramatically improved results by inhibition of mismatch hybridization. Here, we report the use of a CP in the SMAP-2 assay and demonstrate the complete suppression of background amplification, which enables the accurate and rapid genotyping of the UGT1A1*28 microsatellite polymorphism. This is the first report of a CP effectively applied to an isothermal amplification technique.

Materials and Methods

Collection and Preparation of Clinical Specimens

The subjects were 53 healthy Japanese volunteers and 63 cancer patients who received irinotecan-containing chemotherapy in the Yokohama City University Hospital from January 2005 to December 2006. All gave informed consent in writing for use of their peripheral blood in research. Institutional approval for conducting research using human material was obtained from the Ethical Advisory Committee of Yokohama City University School of Medicine and the RIKEN Institute before initiating the study.

All blood samples were collected in a standard EDTA-2Na blood collection tube and stored at -80°C until analyzed. Samples were divided, and one aliquot was used directly for SMAP-2 reactions by diluting the blood approximately 3-fold with 50 mM NaOH and heating at 98°C for 3 min. For each 25-µL SMAP-2 reaction, we added 1 µL diluted and heated blood sample directly to the assay. Genomic DNA was purified from a second aliquot of each sample with the QuickGene-Mini80 kit (Fujifilm, Stamford, CT, USA) and used for PCR sequencing of the UGT1A1 alleles.