INTRODUCTION
Genetic variations that influence pharmacokinetic and pharmacodynamic properties of drugs are now being intro- duced 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 devel- opment 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 misam- plification 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 errone- ously 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 exclu- sively 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, microsat- ellite 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 pharma- cokinetic 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 pharmaco- kinetics of the drug irinotecan and its metabolites (4). We found it difficult to detect this microsatellite polymor- phism 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) (5–7). 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 polymor- phism. This is the first report of a CP effectively applied to an isothermal amplification technique.
Use of a competitive probe in assay design for genotyping of the UGT1A1*28
microsatellite polymorphism by the smart amplification process
Jun Watanabe1,2, Yasumasa Mitani1,3, Yuki Kawai1,3, Takeshi Kikuchi1,3, Yasushi Kogo1,3, Atsuko Oguchi-Katayama1, Hajime Kanamori1,3, Kengo Usui1, Masayoshi Itoh1, Paul E. Cizdziel1, Alexander Lezhava1, Kenji Tatsumi1,2, Yasushi Ichikawa2, Shinji Togo2, Hiroshi Shimada2, and Yoshihide Hayashizaki1,2
1RIKEN, Yokohama, 2Yokohama City University, Yokohama, and 3K.K. DNAFORM, Yokohama, Japan
BioTechniques 43:479-484 (October 2007) 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 polymor- phism. 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.
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.
The SMAP-2 Assay for UGT1A1*28 Microsatellite Polymorphism Detection
SMAP primer sets were designed for amplification and detection of the microsatellite sequences of the UGT1A1 and UGT1A1*28 alleles.
We used FP as a discrimination primer, and FP(Wt) and FP(Mt) were designed to be specific to wild-type allele and mutant allele, respectively. CP was designed to inhibit misannealing of FP primer to the alternative allele template, and the 3′ end of CP was modified by amination (Figure 1).
SMAP-2 reactions were assembled on ice and incubated at 60°C for 60 min. We used the Mx3000P™ Real- Time PCR system (Stratagene, La Jolla, CA, USA) for maintaining isothermal conditions and monitoring the change in fluorescence intensity of interca- lating SYBR® Green I dye (Molecular
Probes™; Invitrogen, Carlsbad, CA, USA) during the reaction. The SMAP-2 method for detecting UGT1A1*28 was carried out in a total reaction volume of 25 μL. Enzymatic components (Aac DNA polymerase and Taq MutS) for the SMAP-2 assays were supplied by K.K. DNAFORM (Yokohama, Japan).
Each reaction contained 2 μM FP and TP, 1 μM boost primer (BP) for TP and BP for FP, 0.25 μM outside primer (OP), 20 μM CP, 1.4 mM dNTPs, 10 mM (NH4)2SO4, 10 mM KCl, 8 mM MgSO4, 1:100,000 diluted original SYBR Green I, 20 mM Tris-HCl, pH 8.8, 0.1% Tween® 20, 12 U Aac DNA polymerase, 1.8 μg Taq MutS, and 1 μL prepared blood sample.
In data analysis and diagnostic judgment, we evaluated the application of SMAP-2 according the principle of amplification versus nonamplifi- cation compared with threshold values.
Generally, we considered amplification to be positive if the fluorescence [Δraw fluorescence (dR): baseline-subtracted fluorescence reading] strength was higher than a value of 1000, and no amplification occurred if the signal was
<1000. Detection times are discussed in this report as the time in which fluorescent values intersected with the intensity value of 1000.
PCR Sequencing of Blood Samples UGT1A1*28 was distinguished from wild-type by sequencing (-147–106) of the PCR product, which yielded a 253- bp product from wild-type sequence and a 255-bp product from the mutant allele. The PCR sequencing method employed was similar to that described previously (8–10).
RESULTS AND DISCUSSION To develop the UGT1A1*28 assay, we first examined real-time ampli- fication profiles of a primer design specific for wild-type allele detection with and without use of a CP. Controlled tests were performed on blood samples known to be homozygous wild-type and homozygous mutant (as determined by PCR sequencing; data not shown). In the absence of CP and without MutS, amplification could be observed in approximately 27 min in homozygous wild-type DNA (Figure 2). Using this same primer set on homozygous UGT1A1*28 mutant DNA, amplifi- cation was evident at approximately 38 min, showing a lag of approximately 11 min for misamplification. By inclusion of a UGT1A1*28 sequence-specific
Figure 1. Allele and smart amplification process version 2 (SMAP-2) primer set sequences for the UGT1A1*28 assay. (A) The genomic sequence of the UGT1A1 promoter region, including the six TA dinucleotide repeats is illustrated. The wild-type UGT1A1 sequence is identical with the exception of six TA dinucleotides instead of seven found in the mutant. (B) The sequence of primers for SMAP-2 assay design is given. In this assay, folding primer (FP) is used as the discrimination primer and FP(Wt) and FP(Mt) differ by only a single dinucleotide. CP(Wt) and CP(Mt) were designed to inhibit mis-anneal- ing of the FP primer to nonspecific alleles and the 3′ end of both CPs were modified by amination. For this assay, only a single outside primer (OP) was used. Although two outside primers are conventional in SMAP, for unknown reasons, use of an additional opposing outside primer in this assay (many were tested) proved inhibitory and consequently was omitted. Wt, wild-type; Mt, mutant; CP, competitive probe; TP, turn-back primer; BP, boost primer.
A
B
CP that harbored seven TA repeats in the assay mix, and by redoing the above experiments on both wild-type and mutant homozygous templates, an effect on both amplification and misam- plification was observed. The amplifi- cation of the wild-type sequence was slightly delayed from approximately 27–36 min, and the misamplification of mutant sequence was delayed from approximately 38–53 min. Although there was an increased lag in misam- plification by inclusion of the CP from
approximately 11–17 min, this effect was not sufficient to support a robust clinical assay. However, a substantial synergistic effect of the CP with Taq MutS was observed and is shown in Figure 3. The inclusion of MutS only had a subtle effect on the conventional SMAP-2 wild-type assay, increasing the misamplification lag from an approximately 11- to 15-min delay. On the other hand, the effect of MutS on the CP-enhanced SMAP-2 wild-type assay was dramatic; full-match ampli-
fication was evident at 45 min, and no misamplification was observed even at 90 min (Figure 3). The design of the reverse mutant-specific SMAP-2 assay for detection of the UGT1A1*28 allele was accomplished using a wild-type homologous CP that harbored six TA repeats in the probe sequence. Similar results to those reported for the wild- type assay were observed. These data indicated that MutS plus CP displayed a synergistic effect in the SMAP-2 assay and were capable of completely suppressing mismatch (background) amplification.
To demonstrate the specificity and utility of the UGT1A1 SMAP-2 assay for handling multiple clinical samples, we examined 116 fresh human blood samples. Detection of both wild-type and mutant alleles for all specimens was evident at approximately 40 min after incubation at 60°C (Figure 4).
No background signal was apparent in any of the assays, indicating that mismatch amplification was completely suppressed. Among the 116 specimens examined by CP-enhanced SMAP-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 validate these results, we also examined all the specimens by PCR sequencing.
The data showed perfect concordance between the SMAP-2 assay results and PCR sequencing (data not shown).
Typical amplification profiles of the CP-enhanced SMAP-2 assays for detection of the UGT1A1 alleles using human blood specimens are shown in Figure 4. 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.
The basic principle of SMAP is that DNA amplification equals detection; no further analysis is required. The basic primer design of SMAP is presented in Supplementary Figure S1 (available online at www.BioTechniques.com). A set of five specially designed primers that recognize a total of six distinct sequences on the target DNA enables the precise amplification of only target-specific sequences. A critical milestone in optimizing SMAP-2 assays is the suppression of exponential
Figure 2. Amplification of UGT1A1 and misamplification of UGT1A1*28 with and without competitive probe (CP). (A) Using a smart amplification process version 2 (SMAP-2) assay design with the folding primer (FP) as the discrimination primer for amplification of the wild-type UGT1A1 allele and no CP, detection of homozygous wild-type DNA from human blood is indicated as the shaded circle (l). Using the same primer set, misamplification of homozygous UGT1A1*28 mutant DNA from human blood is indicated as the shaded square (n). (B) SMAP-2 assay results from using the same prim- er set with a UGT1A1*28-specific CP are also shown. CP slowed the amplification reaction, reduced the yield, and lengthened the lag period of mismatch amplification for approximately 11–17 min. Each graph shows overlaid data from two separate SMAP-2 assays. dR, Δ raw fluorescence.
A B
FP(Wt) – CP + MutS FP(Wt) + CP + MutS
A B
Figure 3. The effect of Thermus aquaticus MutS (Taq MutS) on amplification of UGT1A1 and misamplification of UGT1A1*28 with and without competitive probe (CP). (A) Even when opti- mized, the addition of Taq MutS to the UGT1A1 SMAP-2 assay is not sufficient to abolish misamplifica- tion in the absence of CP. (B) Addition of Taq MutS to the UGT1A1 SMAP-2 assay in the presence of CP resulted in the total suppression of mismatch amplification. Data are shown up to 90 min at which time no mismatch amplification was evident. Each graph shows overlaid data from two separate smart ampli- fication process version 2 (SMAP-2) assays. dR, Δ raw fluorescence.
background amplification. This is usually accomplished by adjusting the design features of the TP, FP, and/or the BP primers and optimization of the concentration of Taq MutS. However, sometimes these approaches are not completely effective, as was the case for detecting the UGT1A1*28 micro- satellite polymorphism due to a high frequency of mismatch amplification.
This phenomenon may be typical of short tandem repeat (STR) polymor- phisms that differ only in copy number of the repeat sequence. To further enhance the suppression of background amplification in a SMAP-2 assay, we developed a further approach using CP.
Use of CP in a SMAP-2 assay to detect a wild-type sequence is illustrated in Figure 5. In the example, the discrimi- nation primer is designed to have full homology to the wild-type allele and can contribute to SMAP amplification because it contains the typical 5′ end design features of FP. On the other hand, the CP is designed to have homology to the mutant sequence and is included in the wild-type assay mix, but cannot contribute to SMAP amplifi- cation because it is a simple primer with no design features for self-priming. By using a CP with complete homology to the repetitive TA dinucleotides and some flanking sequence on either side, hybridization to the mismatch allele can be favored due to its higher melting temperature than the unfavorable mismatch hybridization event to the discrimination primer (FP). The latter event leads to misamplification and generation of background. In cases where mismatch amplification proves to be a persistent issue in SMAP-2 assay development, the addition of a CP may provide an effective approach to enhance assay specificity.
Several different CPs were designed and tested in the development of the UGT1A1*28 SMAP-2 assay (Supplementary Figure S2). Critical elements of its design are that the CP must be a single oligonucleotide that spans across the STR sequence and includes approximately four or more nucleotides on either side. We also found it effective to block the 3′ end of the primer by amination to prevent wasteful participation of the CP in primer extension. Furthermore, there are theoretical limits to the size of the CP as governed by the synthetic
capabilities of primer manufacturers.
Hence STRs of inordinately long sizes may not be addressable by the CP-suppression approach. Perhaps a greater limitation might be the need for significant differential melting temperatures of the full-match CP-target allele versus the CP mismatch allele hybridization event. At the SMAP-2 assay temperature of 60°C, the greater the differential stability of the CP hybridization with exact match target allele versus mismatch allele, the greater the potential for background suppression. CPs that are
too long in size may show little differ- ential stability at 60°C, and thereby function to competitively inhibit the discrimination primer from priming on both templates, resulting only in slower amplification kinetics and not assay specificity enhancement. Finally, the concentration of the CP is also critical, requiring enough to enhance background suppression, but not too much since it slows the overall rate of amplification, presumably in early phases of the SMAP reaction, leading to the generation of key intermediates.
We determined that a concentration of
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Figure 4. Detection of UGT1A1 and UGT1A1*28 alleles by competitive probe (CP)-enhanced smart amplification process version 2 (SMAP-2) assays. The three possible diploid genotypes of the UGT1A1 allele for detection of the UGT1A1*28 microsatellite polymorphism are shown. The left, center, and right graphs are real-time PCR detection of amplification from human blood clinical samples showing homozygous wild-type, heterozygous and a homozygous mutant, respectively. Each graph shows overlaid data from two separate SMAP-2 assays. dR, Δ raw fluorescence.
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Figure 5. Mismatch suppression principle by a competitive probe (CP) on UGT1A1 alleles. For amplification of the wild-type UGT1A1 allele, a discrimination primer [in this case folding primer (FP)]
is designed to hybridize to the region of dinucleotide repeats. (A) Six TA repeats are present in the wild-type allele, and full-match amplification is favored. (B) Mismatch amplification occurs when the wild-type FP hybridizes to a UGT1A1*28 mutant allele (seven TA repeats), resulting in the loop-out of a single AT dinucleotide. (C) A UGT1A1*28 CP has full homology (seven TA repeats), upon hybrid- ization to the UGT1A1*28 mutant allele is capable of blocking mishybridization of the wild-type FP primer. TP, turn-back primer.
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20 μM CP worked well in UGT1A1*28 assay development.
Recently, peptide nucleic acid (PNA) and locked nucleic acid (LNA) polymers were introduced and used in molecular detection assays (11,12).
Completely matched PNA-DNA or LNA-DNA heteroduplex sequences have a higher melting temperature than natural DNA bases, whereas those with a mismatch have a lower melting temperature. In addition, PNA primers cannot participate in extension because they are not recognized by the polymerase. These features are advan- tages that hold great promise for PNA or LNA to become a more effective CP for background suppression in SMAP-2 assay design. However, use of these molecules in clinical assays may be limited due to the high cost and the high concentrations required for each SMAP assay.
Genetic polymorphisms of UDP- glucuronosyltransferase (UGT)1A1, a key metabolizing enzyme of the drug irinotecan (CPT-11), are an important determinant of individual variation in susceptibility to toxicity (10). The UGT1A1*28 polymorphism has particularly been linked to irinotecan- related toxicity by multiple studies (13–17). The FDA has required the package insert of irinotecan to state that UGT1A1*28 is associated with an increased risk of toxicity, and genetic testing is encouraged to decrease this risk.
There have been several previous reports of the allele frequencies of UGT1A1 variants including (TA)5, (TA)6, (TA)7, and (TA)8 in the TATA box in various ethnic groups (18–22).
According to these studies, the (TA)5
and (TA)8 genotypes were not detected in Asians and occurred less frequently in other ethnic groups. Hence, we did not focus on them in this report. But since these microsatellite polymor- phism variants are of a reasonably small size, it is conceivable to develop single oligonucleotides that span the entire length of each repeat and use them as competitive probes in SMAP-2 assays for any or all of these polymorphisms.
Moreover, it is conceivable to apply this method for detecting mononucleotide repetitions, such as Bat-25, Bat-26, and others that are unstable microsat- ellite phenotypic markers in colorectal cancer.
The SMAP method has several key advantages for UGT1A1*28 detection over other genotyping technologies such as PCR sequencing, Pyrosequencing, and the Invader method. The SMAP- 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 purifi- cation (13,23). The SMAP-2 assay is based on strand-displacing DNA polymerase activity and can amplify and detect polymorphisms directly from blood samples, requiring only a simple heat lysis and denaturation step. We could detect the UGT1A1*28 polymor- phism within 50–60 min, including sample preparation time. Compared with other methods, the SMAP-2 assay is relatively inexpensive to employ for genotyping of polymorphisms or mutations. The cost for primers, Aac polymerase, and Taq MutS are probably no more than a few dollars per assay. Less sample prep time and less handling also contribute to lower material and labor costs.
The application of SMAP-2 to molecular diagnostics of UGT1A1*28 has significant potential as evidenced from this study. Sample preparation was extremely minimal, setup was very simple, and detection time was <60 min. The unique primer design, new background suppression technology, and isothermal nature of the assay provide a powerful and unique combi- nation of attributes and a simple protocol for detection of polymor- phisms (including STR) or mutations, making SMAP-2 perhaps the first practical molecular diagnostics tool for bedside usage or point-of-care testing.
ACKNOWLEDGMENTS
We thank K. Nomura (RIKEN) for development of Aac polymerase and Taq MutS. This study was mainly sup- ported by a research grant to the RIKEN Genome Exploration Research Project from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (MEXT) to Y.H.
COMPETING INTERESTS STATEMENT
The authors declare no competing interests.
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Received 11 May 2007; accepted 25 July 2007.
Address correspondence to Yasumasa Mitani, Genome Exploration Research Group, RIKEN Genomic Sciences Center, RIKEN Yokohama Institute, 1-7-22 Suehiro- cho, Tsurumi-ku, Yokohama, Kanagawa, 230-0045, Japan. e-mail: mitani@gsc.
riken.jp; and Jun Watanabe, Department of Gastroenterological Surgery, Yokohama City University Graduate School of Medicine, 3-9 Fukuura, Kanazawa-ku, Yokohama, Kanagawa 236-0004, Japan.
e-mail: [email protected]
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