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Adaptation of SmartAmp-2 Assays for Microsatellite Copy Number Polymorphism Detection on the LightCycler^® 480 Instrument

Jun Watanabe^1,2, Yasumasa Mitani^1,3, Alexander Lezhava¹, and Yoshihide Hayashizaki<sup>1,2

1</sup>RIKEN, Yokohama, Japan, ²Yokohama City University, Yokohama, Japan, ³K.K. DNAFORM, Yokohama, Japan

Corresponding author

Introduction

The Smart Amplification process, version 2 (SMAP-2), also known as SmartAmp-2, is a rapid polymorphism detection system based on a unique asymmetric primer design, including a folding primer (FP) and a turn-back primer (TP) [1]. Combined with Thermus aquaticus MutS (Taq MutS) protein in an isothermal amplification procedure, the technique enables suppression of mis-amplification of highly related sequences (e.g., a wild-type sequence versus an SNP variant), minimizing background amplification. The Taq MutS protein binds to mis-matched nucleotides in erroneously primed dsDNA and prevents further amplification by blocking the dissociation of  DNA duplexes having a mis-match by DNA polymerases having strand displacement activity. SmartAmp-2 assays have been used for detection of various SNPs and mutations, e.g., in ALDH2 and EGFR genes [1,2]. In the study presented here, we investigated whether this assay principle could be adapted for detection of microsatellite polymorphisms showing copy number differences in 2–4 nucleotide repeats, using the UGT1A1*28 polymorphism as an example. The TATA box in the UGT1A1 promoter 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 [3].

In previous reports, the assay specificities of allele-specific PCR, oligonucleotide microarrays, and other techniques were shown to be enhanced with the use of a competitive probe (CP) [4–6]. It was reported that addition of a CP dramatically improved results by inhibition of mis-match hybridization. Here we report the use of a CP in the SmartAmp-2 assay and demonstrate the complete suppression of background amplification, which enables the accurate and rapid genotyping of the UGT1A1*28 microsatellite polymorphism.

The basic principle of SmartAmp is that “DNA amplifica-tion equals detection”, due to a set of specially designed primers that recognize distinct sequences on the target DNA enabling the precise amplification of only the target-specific sequences. To further enhance the suppression of background amplification in a SmartAmp-2 assay, we developed another approach using a competitive probe (CP). The use of a CP in a SmartAmp-2 assay to detect a wild-type sequence is illustrated in Figure 1. The discrimination primer is designed to have full homology to the wild-type allele and can contribute to SmartAmp amplification because it contains the typical 3’ end design features of a FP. The CP is designed to have homology to the mutant sequence and is included in the wild-type assay mix, but unlike TP or FP it cannot contribute to SmartAmp amplification because it is a simple primer with no design features for self-priming. In addition, its 3’ end is blocked by amination to prevent wasteful participation in primer extension. By using a CP with complete homology to the repetitive “TA” dinucleotides with some flanking sequence on either side, hybridization to the mis-match allele can be favored due to its higher melting temperature (Tm) compared with the unfavorable mis-match hybridization event of the discrimination primer (in this case an FP). The latter event leads to mis-amplification and generation of background.                

In cases where mis-match amplification proves to be a persistent issue in SmartAmp-2 assay development, the addition of a CP may provide an effective approach to enhancing assay specificity.

Materials and Methods

Collection and preparation of research samples

Research samples were obtained from 53 healthy Japanese volunteers and 63 individuals with oncological disorders. All gave informed consent in writing for use of their peripheral blood in research.

The SmartAmp-2 assay for UGT1A1*28
microsatellite polymorphism detection

Primers for SmartAmp-2 assays were designed for amplification and detection of the microsatellite sequences of the UGT1A1*28 alleles as described in a previous report [7]. Assays described previously were transferred successfully to the LightCycler^® 480 System by maintaining isothermal conditions and monitoring the change in fluorescence intensity of intercalating SYBR^® Green I dye during the reaction. We evaluated the application of SmartAmp-2 according to the principle of amplification versus non-amplification compared with threshold values.

Results and Discussion

The design of the reverse mutant–specific SmartAmp-2 assay for detection of the UGT1A1*28 allele was accomplished using a wild-type homologous competitive probe (CP) which harbored six “TA” repeats in the probe sequence. Detection of both wild-type and mutant alleles for all specimens were evident at ~40 min after incubation at 60°C (Figure 2). No background signal was apparent in any of the assays, indicating that mis-match amplification was completely suppressed. Among the 116 specimens examined by CP-enhanced SmartAmp-2 assays for the UGT1A1 wild-type and *28 alleles, 90 tested homozygous for wild-type, 25 were heterozygous and 1 was homozygous mutant. To confirm these results, we also examined all specimens by PCR-sequencing. The data showed perfect concordance between the SmartAmp-2 assay results and PCR-sequencing (data not shown).

Typical amplification profiles of the CP-enhanced SmartAmp-2 assays for detection of the UGT1A1 alleles using human blood specimens are shown in Figure 2. Each graph is a composite of two assays performed on a single specimen: a UGT1A1 wild-type allele assay, and a UGT1A1*28 mutant allele assay. Representative data from the three possible diploid genotypes are shown.

We found that by the addition of a discriminating primer acting as a CP to the amplification reaction, assay speci-ficity could be significantly enhanced. Including sample preparation time and use of a CP-enhanced SmartAmp-2 assay, we rapidly detected the UGT1A1*28 polymorphism within 60 minutes.

The SmartAmp method has several key advantages for UGT1A1*28 detection over other genotyping technologies such as PCR-sequencing or pyrosequencing. The SmartAmp-2 technique is simple, requires no DNA purification and is performed in a closed tube, which reduces the risk of contamination. Other methods generally require careful DNA purification. The SmartAmp-2 assay uses DNA polymerases having a strand-displacing activity, and it can amplify and detect polymorphisms directly from blood samples requiring only a simple heat lysis and denaturation step. We were able to detect the UGT1A1*28 polymorphism within 60 minutes including sample preparation time. Compared with other methods, the SmartAmp-2 assay is a relatively inexpensive way to carry out genotyping of polymorphisms and mutations. The cost of primers, Aac polymerase, and Taq MutS  is probably no more than a few dollars per assay. Less sample preparation time and less handling also contribute to lower material and labor costs.          

References

1. Mitani Y et al. (2007) Nat Methods 4:257–262
2. Kanako H et al. (2007) Cancer Res, in press
3. Hasegawa Y et al. (2006) Ann NY Acad Sci 1086:223–232
4. Park JH et al. (2004) Clin Chem 50:1688–1691
5. Parsons BL et al. (2005) Methods Mol Biol 291:235–245
6. Gibbs RA et al. (1989) Nucleic Acids Res 17:2437–2448
7. Watanabe JY et al. (2007) Biotechniques 43:479–484
   

This article was originally published in Biochemica 3/2009, pages 28-30. ©Springer Medizin Verlag 2009