Rolling Circle Amplification Combined with Gold Nanoparticle

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Rolling Circle Amplification Combined with Gold Nanoparticle Aggregates for Highly Sensitive Identification of Single-Nucleotide Polymorphisms Jishan Li,† Ting Deng,†,‡ Xia Chu,*,† Ronghua Yang,*,† Jianhui Jiang,† Guoli Shen,† and Ruqin Yu† State Key Laboratory of Chem/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University, Changsha, 410082, China, and Institute of Biological and Environmental Science & Technology, Central South University of Forestry and Technology, Changsha, 410004, China A highly sensitive and specific colorimetry-based rolling circle amplification (RCA) assay method for single-nucleotide polymorphism genotyping has been developed. A circular template is generated by ligation upon the recognition of a point mutation on DNA targets. An RCA amplification is then initiated using the circular template in the presence of Phi29 polymerase. The RCA product can be digested by a restricting endonuclease, and the cleaved DNA fragments can mediate the aggregation of gold nanoparticle-tagged DNA probes. This causes a colorimetric change of the solution as the indicator of the mutation occurrence, which can be detected using UV-vis spectroscopy or viewed by naked eyes. On the basis of the high amplification efficiency of Phi29 polymerase, a mutated target of ∼70 fM can be detected in this assay. In addition, the protection of the circle template using phosphorothioated nucleotides allows the digestion reaction to be performed simultaneously in RCA. Moreover, DNA ligase offers high fidelity in distinguishing the mismatched bases at the ligation site, resulting in positive detection of mutant targets even when the ratio of the wildtype to the mutant is 10 000:1. The developed RCA-based colorimetric detection scheme was demonstrated for SNP typing of β-thalassemia gene at position -28 in genomic DNA. Single nucleotide polymorphisms (SNPs) are the most common variations in human genomes and may be responsible for the phenotypic differences among individuals.1 Studies have found close associations between SNP and tumor development or progression, and analysis of polymorphisms can provide a tool for early diagnosis and risk assessment of malignancy.1–3 Recent advancements in biotechnologies have offered a wide variety of choices for SNP detections. In principle, these approaches can be categorized into three kinds. One design type is based on the * To whom correspondence should be addressed. E-mail: [email protected] (X.C.); [email protected] (R.Y.). Tel.: 86+731-8882 2523. Fax: 86+731-8882 2523. † Hunan University. ‡ Central South University of Forestry and Technology. (1) Engle, L. J.; Simpson, C. L.; Landers, J. E. Oncogene 2006, 25, 1594–1601. (2) Carlton, V. E. H.; Ireland, J. S.; Useche, F.; Faham, M. Human Genomics 2006, 2, 391–402. (3) McCarthy, J. J.; Hilfiker, R. Nat. Biotechnol. 2000, 18, 505–508. 10.1021/ac100336n  2010 American Chemical Society Published on Web 03/01/2010

single-base mismatch induced difference in local chemical environments or secondary structures.4-6 Another strategy is to inspect the thermodynamic properties, such as hybridization free energy7,8 and melting temperature9-11 of the hybridized product, via precise temperature control. The third kind method is built on the enzymatic reactions such as allele-specific extension by polymerase,12,13 sequence or conformation specific cleavage by endonuclease14,15 and allele-specific ligation by ligase.16-18 While each strategy has its distinct advantages, each also presents a unique set of limitations with respect to simplicity, sensitivity, ease of multiplexing, and throughput, etc. Continuous development of alternative SNP screening technologies is still underway. Rolling circle amplification (RCA), an isothermal DNA replication technique, has been proven very useful for highly sensitive detection of target nucleic acids and proteins.19,20 In RCA-based assays, target quantification is achieved through the quantification of the RCA products. Different techniques have been utilized for this purpose. For instance, for amplifications performed on solid surfaces, the RCA products hybridized to multiple oligonucleotide fluorescent probes, and the fluorescence was measured by a conventional microarray scanning device.21–23 For solution-based (4) Okamoto, A.; Tainaka, K.; Saito, I. J. Am. Chem. Soc. 2003, 125, 4972– 4973. (5) Okamoto, A.; Tanaka, K.; Fukuta, T.; Saito, I. J. Am. Chem. Soc. 2003, 125, 9296–9297. (6) Masato, O.; Hiroyuki, I.; Hiroshi, K.; Kenshi, H.; Takao, S. Proc. Natl. Acad. Sci. U.S.A. 1989, 86, 2766–2770. (7) Tyagi, S.; Kramer, F. R. Nat. Biotechnol. 1996, 14, 303–308. (8) Wei, F.; Chen, C.; Zhai, L.; Zhang, N.; Zhao, X. S. J. Am. Chem. Soc. 2005, 127, 5306–5307. (9) Gerion, D.; Chen, F.; Kannan, B.; Fu, A.; Parak, W. J.; Chen, D. J.; Majumdar, A.; Alivisatos, A. P. Anal. Chem. 2003, 75, 4766–4772. (10) Russom, A.; Haasl, S.; Brookes, A. J.; Andersson, H.; Stemme, G. Anal. Chem. 2006, 78, 2220–2225. (11) Ross, P. L.; Lee, K.; Belgrader, P. Anal. Chem. 1997, 69, 4197–4202. (12) Vreeland, W. N.; Meagher, R. J.; Barron, A. E. Anal. Chem. 2002, 74, 4328– 4333. (13) Chen, X.; Kwok, Y. P. Nucleic Acids Res. 1997, 25, 347–353. (14) Ross, P.; Hall, L.; Smirnov, I.; Haff, L. Nat. Biotechnol. 1998, 16, 1347– 1351. (15) Nie, B.; Shortreed, M. R.; Smith, L. M. Anal. Chem. 2005, 77, 6594–6600. (16) Tobe, V. O.; Taylor, S. L.; Nickerson, D. A. Nucleic Acids Res. 1996, 24, 3728–3732. (17) Landegren, U.; Kaiser, R.; Sanders, J.; Hood, L. Science 1988, 241, 1077– 1080. (18) Barany, F. Proc. Natl Acad. Sci. USA 1991, 88, 189–193. (19) Fire, A.; Xu, S. Proc. Natl. Acad. Sci. U.S.A. 1995, 92, 4641–4645. (20) Schweitzer, B.; Wiltshire, S.; Lambert, J.; O’Malley, S.; Kukanskis, K.; Zhu, Z.; Kingsmore, S. F.; Lizardi, P. M.; Ward, D. C. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 10113–10119.

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Figure 1. Schematic illustration of the RCA reaction and AuNP assembly-based assay: (1) hybridization between padlock probe and target and ligation; (2) RCA and digestion; (3) colorimetric detection via the assembly of AuNP-tagged DNA probes.

reactions, a confocal microscope or a flow cytometer was used to count individual RCA products.24-27 These approaches need to use complex labeling or surface functionalization chemistry and usually require expensive measurement instrumentation. Recently, Zhong’s group combined RCA with electrophoresis to realize the identification of DNA or RNA targets.28,29 However, these methods required multiple treatment processes and sophisticated instrumentation. At the same time, focusing on the development of more simple and rapid bioassay methods, gold nanoparticles (AuNPs) have emerged due to their unique size-dependent surface plasmon resonance absorption.30,31 Recently, DNA-functionalized AuNPs have been used for both chemical and biological detections based on the discriminated effects of different DNA structures on the aggregations of AuNPs.32-35 Although these approaches have the advantage of easily read out with naked eye, they are limited in application for SNP detection due to the limited sensitivity of colorimetry. Specifically, the incorporation of RCA technology with (21) Gerry, N. P.; Witowaski, N. E.; Day, J.; Hammer, R. P.; Barany, G.; Barany, F. J. Mol. Biol. 1999, 292, 251–262. (22) Nallur, G.; Luo, C.; Fang, L.; Cooley, S.; Dave, V.; Lambert, J.; Kukanskis, K.; Kingsmore, S.; Lasken, R.; Schweitze, B. Nucleic Acids Res. 2001, 29, e118. (23) Schweitzer, B.; Roberts, S.; Grimwade, B.; Shao, W.; Wang, M.; Fu, Q.; Shu, Q.; Laroche, I.; Zhou, Z.; Tchernev, V. T.; Kingsmore, S. F. Nat. Biotechnol. 2002, 20, 359–365. (24) Li, M.; Diehl, F.; Dressman, D.; Vogelstein, B.; Kinzler, K. W. Nat. Methods 2006, 3, 95–97. (25) Jarvius, J.; Melin, J.; Go ¨ransson, J.; Stenberg, J.; Fredriksson, S.; GonzalezRey, C.; Bertilsson, S.; Nilsson, M. Nat. Methods 2006, 3, 725–727. (26) Blab, G. A.; Schmidt, T.; Nilsson, M. Anal. Chem. 2004, 76, 495–498. (27) Li, J. S.; Zhong, W. Anal. Chem. 2007, 79, 9030–9038. (28) Li, N.; Li, J. S.; Zhong, W. Electrophoresis 2008, 29, 424–432. (29) Li, N.; Jablonowski, C.; Jin, H. L.; Zhong, W. Anal. Chem. 2009, 81, 4906– 4913. (30) Mirkin, C. A.; Letsinger, R. L.; Mucic, R. C.; Storhoff, J. J. Nature 1996, 382, 607–609. (31) Elghanian, R.; Storhoff, J. J.; Mucic, R. C.; Letsinger, R. L.; Mirkin, C. A. Science 1997, 277, 1078–1081. (32) Li, H.; Rothberg, L. Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 14036–14039.

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AuNPs-based colorimetric detection will combine the high sensitivity of RCA with the convenience and aesthetic appeal of a visual assay.36 However, no effort has been made for this attempt because the RCA products are large, megabase fragments and fold into micrometer-sized random coils in solution, which are not suitable for the AuNP-tagged probe hybridization. In this paper, we attempt to develop a rapid, sensitive, and specific SNP detection method for point mutation in clinical diagnosis through the combination of RCA and AuNP-based colorimetric assay. Figure 1 illustrates the scheme for SNP detection. The 3′- and 5′-terminal sequences of the padlock probe are first hybridized on the target. Then, the two termini are joined by a DNA ligase37 when the base at the 3′-end of the padlock probe is perfectly matched to the DNA target. Only for the ligated padlock probe, the RCA is initiated to generate a product that is simultaneously digested into identical fragments by a restriction endonuclease. Hybridization of the DNA fragments thus produced with AuNP-tagged probes results in the formation of a polymeric AuNP-polynucleotide aggregate, triggering a red to purple color change in the solution. Then, the point mutation is discriminated via the visual colorimetry. The proposed approach is demonstrated using a model system for the identification of point mutations in β-thalassemia gene at position -28 (AAA mutated to AGA). This mutation is one of the most commonly met mutations among the population in the southwest of China. The results revealed that both the wild-type and mutant were successfully scored. (33) Li, J. S.; Chu, X.; Liu, Y.; Jiang, J. H.; He, Z.; Zhang, Z.; Shen, G.; Yu, R. Q. Nucleic Acid Res. 2005, 33, e168. (34) Wang, J.; Wang, L. H.; Liu, X. F.; Liang, Z. Q.; Song, S. P.; Li, W. X.; Li, G. X. Adv. Mater. 2007, 19, 3943–3946. (35) Wang, H.; Wang, Y. X.; Jin, J. Y.; Yang, R. H. Anal. Chem. 2008, 80, 9021– 9028. (36) Baner, J.; Nilsson, M.; Mendel-Hartvig, M.; Landegren, U. Nucleic Acid Res. 1998, 26, 5073–5078. (37) Nilsson, M.; Malmgren, H.; Samiotaki, M.; Kwiatkowski, M.; Chowdhary, B. P.; Landegren, U. Science 1994, 265, 2085–2088.

Table 1. Oligonucleotides Synthesized in this Experimenta P1 P2 P3 T1 T2 RCA primer Dp1 Dp2

5′-pTATGCCCAGCCCTG taaga tgaagata gcgcac aatggtcggattctcaactcgtaTCTGCCCTG ACTTC-3′ 5′-TATGCCCAGCCCTG taaga tgaagata gcgcac aatggtcggattctcaactcgtaTCTGCCCTG ACTTC-3′ 5′-pAAGTCAGGGCAGAG taaga tgaagata gcgcac aatggtcggattctcaactcgtaAGGGCTGG GCATAA-3′ 5′-cagggctgggcatagaagtcagggcagag-3′ 5′-cagggctgggcataaaagtcagggcagag-3′ 5′-tgcgctatcttca-3′ 5′-(SH)-(CH2)6-caa tgg tcg gat-3′ 5′-tct caa ctc gta-(CH2)3-(SH)-3′

a The padlock probes were designed with the help of DNA probe design software (Zucker folding program, http://www.bioinfo.rpi.edu/ applications/mfold/old/dna/form1.cgi). The designed DNA sequences were used for detection of point mutation in position -28 of the β-thalassemia gene. For the particular mutation, the fifth position base A of the sequence CATAAA (TATA box) mutates to G, which affects the transcriptional activity of β-globin. The bold base in p1 and p3 means that these bases were phosphorothioates, and “p” represents a phosphate at 5′. The circled base in T1 indicates the mutant base.

EXPERIMENTAL SECTION Oligonucleotides and Chemicals. Oligonucleotides (Table 1), E. coli DNA ligase, HhaI restriction enzyme, albumin from bovine serum (BSA), Taq DNA polymerase, and the mixture of deoxyribonucleotides (dNTPs) were all obtained from Takara Biotechnology Co., Ltd. (Dalian, China), and were used as received. RepliPHI Phi29 DNA polymerase was purchased from Epicenter (Madison, WI). Nicotinamide adenine dinucleotide (oxidized, NAD+) was from ICN (Germany). Other chemicals were all purchased from Amresco (Solon, OH). Deionized water was obtained through a Nanopure Infinity ultrapure water system (Barnstead/thermolyne Corp, Dubuque, IA) and had an electric resistance >18.2 MΩ. AuNPs of ca. 13-nm diameter were prepared according to documented procedures.38 The preparation of 3′- or 5′-(alkanethiol) oligonucleotidemodified Au nanoparticles (Dp1, Dp2) was performed as reported previously.33 The colloids were redispersed in a 0.3 M KCl-0.1% (v/v) Triton X-100-30 mM Tris-HCl (pH 7.8) solution and stored in a refrigerator at 4 °C. Assay Process. In a typical experiment, first, a 40-µL reaction mixture in solution (30 mM pH 7.8 Tris-HCl, 0.1 M KCl, 0.1 mM NAD+, 4 mM MgCl2), which contained 162 nM of padlock probe and a certain target oligonucleotide, was heated up to about 95 °C and then cooled down to room temperature (For genomic DNA assay, the denatured mixture was immersed in ice-water immediately.), BSA (final concentration is 0.05%) and E. coli DNA ligase (final concentration is 0.032 U/L) were added. Then, the reaction mixture was incubated in a 37 °C water bath for 1 h. Second, the RCA and cleavage reaction was carried out with the addition of 1.7 µL 10 µM primer solution, 1.6 µL 0.2 M MgCl2, 6.4 µL 25 mM dNTPs, 6.4 µL 50 mM TrisHCl (pH 7.5), 2 µL 100 U/µL Phi29 DNA polymerase, 0.5 µL 10 µg/µL acetylated BSA, and 1.3 µL 10 U/µL HhaI to the reaction mixture followed by incubation at 37 °C for 2 h. Third, 1 µL of 1 M trisodium citrate and certain AuNp-tagged probes solution (Dp1 and Dp2) were added to perform a 30 min (38) Storhoff, J. J.; Elghanian, R.; Mucic, R. C.; Mirkin, C. A.; Letsinger, R. L. J. Am. Chem. Soc. 1998, 120, 1959–1964.

hybridization at 37 °C. Then colorimetric or UV-vis (Mutispec1501 Shimadzu Hyper UV, Japan) analysis was performed. PCR Amplification of Genomic DNA. PCR amplification was performed in 50 µL of 10 mM Tris-HCl buffer (pH 8.3) with 10 mM KCl, 4.0 mM MgCl2, 250 µM dNTPs, 1 µM forward and reverse primers (50 pmol for each primer), and about 40 ng of genomic DNA extracted from the patient’s blood cells. For generation of a 207 bp PCR product, the following primers were used: (forward) 5′-aatctactcccaggagcagg-3′; (reverse) 3′-ctacttcaaccaccactccg-5′. Amplification was achieved by thermal cycling for 40 cycles at 95 °C for 30 s, 60 °C for 30 s, 72 °C for 1 min, and a final extension at 72 °C for 10 min. PCR products were purified by the ethanol precipitation method and redissolved in 50 µL of deionized water. RESULTS AND DISCUSSION Design of the Detection Scheme. Normally, the padlock probe was hybridized onto a target and formed a circular probe by the E. coli DNA ligase if it was a perfect match between them, which was then amplified by the replication primer and Phi29 polymerase. By measuring RCA product, the specific DNA target could be identified. However, due to RCA products that were very large and megabase fragments, the formed gold nanopaticle aggregate folded into millimeter-sized random cross-linking coils in solution (Supporting Information (SI), Figure S1), making it difficult to perform quantification of RCA. Since the RCA product was a single-strand DNA fragment with repeated sequences arranged end to end, it can be digested into identical fragments by restriction enzymes if a digestion site was incorporated to the template sequence. In the present study, a sequence of 5′-GCGC3′ that can be recognized by HhaI was introduced to the padlock probe to produce the cutting-sites (5′-GCG∧C-3′) on RCA products.28 At the same time, in order to maintain a continuous RCA, the circle template must not be cleaved during the digestion of RCA products. So, the phosphorothioate modification, often used in the strand displacement amplification reaction,39,40 was used to protect the circle template from cutting by the restriction endonuclease. This design enables us to perform RCA and the enzyme digesting reaction simultaneously. In the present approach, HhaI not only works within the double-stranded region but also cuts the single-stranded region via the transiently formed double-stranded hairpin structures at a relatively fast reaction speed compared to other endonucleases like CfoI.41 The characteristic of HhaI was investigated through sol-gel electrophoresis (SI, Figure S2). The result shows that the padlock probes without phosphorothioates (P2, Table 1) can be cleaved into smaller DNA fragments (about 30 and 40 bp) by the HhaI, while the phosphorothioated padlock probe (P1, Table 1) cannot. The result also indicates that the RCA product can be digested into small fragments with a length equaling to that of probe 1. After RCA and the enzyme digestion reactions, the AuNP-tagged probe was added into the reaction solution. As shown in Figure 2, the AuNP-tagged probe in the blank solution exhibits the nanoparticle’s characteristic surface plasma absorption peak at 520 nm and appears pink-red. The spectrum for the mismatch target (T2) also (39) Dean, F. B.; Nelson, J. R.; Giesler, T. L.; Lasken, R. S. Genome Res. 2001, 11, 1095–1099. (40) Giusto, D. D.; King, G. C. Nucleic Acid Res. 2003, 31, e7. (41) Reckmann, B.; Krauss, G. Biochim. Biophys. Acta 1987, 908, 90–96.

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Figure 3. Effect of time on the ligation efficiency. Hybridization times of 10, 30, 60, and 90 min between padlock probe (P1, 162 nM) and its perfect-matched target (T1, 100 nM) were choosed to investigate the ligation efficiency. Other experimental conditions were described as in the Experimental Section.

Figure 2. (A) UV-vis absorption spectra of the AuNP-tagged detection probes (Dp1 and Dp2) after incubation with the reaction mixture without target (a) and with mismatched target T2 (b) or perfectly matched target T1 (c). (B) Corresponding color change of the reaction mixture. The concentration of the padlock is 162 nM, and the concentrations of T1 and T2 are 100 nM, respectively.

shows the characteristic of separate AuNPs, and the solution color is still red. In contrast, for a perfectly matched target (T1), the surface plasma absorption peak was shifted to long wavelengths with a concomitant color change of the solution from pink-red to purple, even to gray, due to the hybridization between the AuNPtagged probe and the products from RCA and enzyme digestion. This implies that the nick between the two adjacent probes was sealed and the RCA was performed for the perfectly matched target, while not for the mismatched, demonstrating that the developed method holds promise for SNP detection. Optimization of the Reaction Conditions. Since the effect of external factors on the ligation had been previously reported,42,43 here only the hybridization time between the padlock probe and the target was investigated. To quantitatively estimate the effect of reaction time on the efficiency in generating circular probes in the ligase reaction, after ligation the RCA and enzyme digestion was carried out followd by the addition of AuNP-tagged probes. Then, the effect of reaction time on the efficiency in generating circular probes could be estimated from the absorbance change of the reaction mixture at 650 nm. It was observed from Figure 3 that the absorbance at 650 nm was no longer increased after the hybridization time between the padlock probe and target was over (42) Li, J. S.; Chu, X.; Jiang, J. H.; Shen, G.; Yu, R. Q. Analyst 2008, 133, 939– 945. (43) Liu, L.; Tang, Z. W.; Wang, K. M.; Tan, W. H.; Li, J.; Guo, Q.; Meng, X.; Ma, C. B. Analyst 2005, 130, 350–357.

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Figure 4. Effect of time on the yield of RCA products. RCA durations of 10, 30, 60, 90, 120, and 150 min of RCA duration were selected to investigate the yield of RCA products. The concentration of padlock probe (P1) and its perfect-matched target (T1) is 162 nM and 100 nM, respectively. Other experimental conditions were described as in the Experimental Section.

60 min, indicating that the formed circular probe has reached saturation when the hybridization time was over than that time. In our experiment, 60 min was selected as the incubation time for circular probe formation. In order to obtain higher sensitivity, the RCA duration was investigated. The results are shown in Figure 4. Under a higher concentration of phi29 polymerase and substrates, the optimal RCA duration was found to be at about 2 h from the plot of absorbance change at 650 nm versus RCA duration. RCA duration longer than 2 h actually did not yield significant increase for the product signal. This might be due to the fact that the RCA reaction had reached equilibrium and the activity of phi29 polymerase was substantially inhibited by the RCA products after 2 h. So 2 h was chosen as the RCA and enzyme digestion time. Sensitivity of Detection. Next, we implemented this RCAbased colorimetric assay to detect and quantify the synthetic DNA targets. It was observed from Figure 5 that the absorbance change versus the logarithm of target concentration showed very good linearity in the range from 0.3 pM to 80 pM. The detection limit, which was calculated in terms of three times the signal-to-noise level, is ca. 70 fM. This detection limit was several hundred-fold lower than that of the conventional AuNPs aggregate-based

Figure 5. Quantitative analysis of the target DNA T1 using UV-vis spectrophotometry. The experimental condition was described in the Experimental Section. The standard deviations obtained by three repeated measurements were shown as the error bars.

colorimetric DNA assay.33,44 Moreover, when the concentration of the target was more than 1 pM, an obvious color change could be discriminated by visualization. A relatively narrow dynamic range of 2 orders of magnitudes was obtained in our study, because a higher target amount (over 80 pM) would saturate the AuNP-tagged probes. Specificity Study. To evaluate an SNP detection assay, specificity was one of the most important factors, because it reflected the ability of the assay to avoid false negative or false positive readouts.45 Excellent performance in this regard was required before adapting the assay to clinical applications. The specificity of our RCA-based colorimetric assay was determined by the fidelity of the DNA ligase. Among common DNA ligases commercially available, Tth and E. coli ligases were proved to be more sensitive to mismatches than T4 ligase.43,46,47 However, the working temperature of Tth ligase (65 °C) was much higher than the melting temperatures of our padlock probes (around 50 °C). Instead, E. coli ligase had a working temperature of 37 °C and was thus employed in the present study. The other reason for choosing E. coli ligase was that, the optimal working temperature (37 °C) was suitable for combining the hybridization process together with the ligation process as well as the RCA reaction. To assess the specificity of our assay, we mixed oligonucleotides T2 and T1 in a total concentration of 100 nM at different mole ratios of 10 000:1, 5000:1, 2000:1, 499:1, 99:1, and 9:1. These two oligonucleotides represented the wild-type (T2) and the mutant (T1) targets of -28 β-thalassemia gene. The blank reaction contained only the wild-type target (T2) of 100 nM. The padlock probe of P1 (100 nM) with sequence perfectly complementary to the mutant target was added to the reaction. The results are displayed in Figure 6. In the control experiment using the wildtype target T2, we obtained a slightly increased absorbance at 650 nm. This might be corresponding to the aggregation of AuNPtagged probes resulting from the low-efficiency ligation of mismatched padloack probes. However, when the perfect-matched target was present, even if the ratio of the wild-type target to the mutant is 10 000:1 (10 pM T1 in the mixture), the absorbance (44) Reynolds, R. A.; Mirkin, C. A.; Letsinger, R. L. J. Am. Chem. Soc. 2000, 122, 3795–3796. (45) Bjorheim, J.; Ekstrom, P. O. Electrophoresis 2005, 26, 2520–2530. (46) Luo, J.; Bergstrom, D. E.; Barany, F. Nucleic Acids Res. 1996, 24, 3071– 3078. (47) Tong, J.; Cao, W.; Barany, F. Nucleic Acids Res. 1999, 27, 788–794.

Figure 6. Histogram showing the capacity to measure the perfectmatched target in a mixture of T1 and T2. The concentration of padlock probe is 100 nM, and the total target concentration (T1 + T2) also is 100 nM. The detailed experimental condition was described in the Experimental Section. The standard deviations obtained by three repeated measurements are shown as the error bars. Table 2. Analysis Results of Real Gene Samplesa

sample

P1 for mutant target color change ?

P3 for normal target color change ?

gene type

1 2 3 4 5 6 7

+ + + +

+ + + + + + +

N/N -28/N N/N N/N -28/N -28/N -28/N

a N/N, -28/N, or -28/-28 means homozygous wild-type, heterozygous mutant, and homozygous mutant, respectively. The symbol “+” means that the color of solution has a change from pink-red to blue-gray, while, “-” means that the solution has no color change. For each RCA assay, 2 µL of PCR product solution was used.

increase was still significant and higher than that for the control experiment using 100 nM wild-type target. Furthermore, we observed that the absorbance increment was linearly correlated to the concentration of the perfectly matched target T1. This result demonstrated the extraordinary capability of our RCA-based colorimetric assay in detecting a low-abundance mutation in the presence of a large quantity of the wild-types. This capability, along with its high sensitivity and simplicity, granted the assay great potential in clinical applications. Analysis of Genomic DNA. After proving the specificity and sensitivity of our stand-alone RCA-colorimetric assay, we applied it to detecting the PCR products of real sample β-thalassemia genomic DNA under the aforementioned optimized conditions. In order to evaluate the ability of the developed method in typing the genomic sample as homozygous or heterozygous type, a second probe with complementary sequence to the wild-type sequence was designed (P3, Table 1). If the color of the solution changed to purple for P1 while not for P3, one could easily realize that the sample was homozygous mutant. On the contrary, if the solution color changed to purple for P3 while not for P1, the sample could be identified as homozygous wild-type. However, if the solutions color changed to purple for both P1 and P3, the sample could be heterozygous mutant. With this in mind, we could analyze the PCR products of different genomic samples by using Analytical Chemistry, Vol. 82, No. 7, April 1, 2010

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these two probes. The results are shown in Table 2. One observed that, when P1 was used, the color of solutions showed appreciable changes from pink-red to blue-gray for samples of 2, 5, 6, and 7, while no remarkable color changes were obtained for samples of 1, 3, and 4. This suggested that there was no point mutation at the -28 site in samples 1, 3, and 4, while a point mutation was present in samples 2, 5, 6, and 7. When P3 was used, the color of solutions changed to blue-gray for samples 1-7, indicating that the wild-type target was present in all the samples. So, the samples 1, 3, and 4 were identified as homozygous wild-type, while the samples 2, 5, 6, and 7 were heterozygous mutant. No homozygous mutant type was found in these samples. These results were further verified by the sequencing data, from which we identified that the base at -28 site was “A” for samples 1, 3, and 4, and for samples 2, 5, 6, and 7 the base was “G” and A. These results demonstrated that the developed method could be successfully used for SNP detection of real genomic DNA samples. CONCLUSION The present study proposed a new colorimetric approach for simple and sensitive detection and quantification of target gene containing SNP. Neither complicated fluorescent or radioisotopelabeled technique nor sophisticated instruments were needed in the assay, and it took less than 4 h to finish an assay. The principle of this method was based on the combination of the high-fidelity ligation of DNA ligase and signal enhancement of RCA with the simple yet powerful colorimetric detection of AuNPs assembly.

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The RCA-based colorimetric assay was a sensitive, low cost, and simple technique for nucleic acid detection. Compared with the nanomolar detection limit of traditional AuNP-based colorimetric DNA assay, the present method could achieve a detection limit as low as 70 fM through three simple treatment steps: ligation, RCA and enzyme digestion, and colorimetric assay. In addition, the remarkable fidelity of DNA ligase in distinguishing the mismatches allowed the detection of low-abundance mutant in the presence of 10 000 times more wild-type targets. This new RCAbased colorimetric method might prove useful in clinical diagnosis of genetic diseases that contained single nucleotide mutations. ACKNOWLEDGMENT This work was supported by “973” National Key Basic Research Program (2007CB 310500) and National Natural Science Foundation of China (20775005, 20875027, 20975035) and Hunan Province (07JJ1002). J.L. also thanks Hunan University for the start-up funds (521105668). SUPPORTING INFORMATION AVAILABLE Figures S1 and S2. This material is available free of charge via the Internet at http://pubs.acs.org.

Received for review December 6, 2009. Accepted February 15, 2010. AC100336N