Published on Web 08/03/2007
Electrochemical Quantification of Single-Nucleotide Polymorphisms Using Nanoparticle Probes Guodong Liu and Yuehe Lin* Contribution from Pacific Northwest National Laboratory, Richland, Washington 99352 Received January 19, 2007; E-mail:
[email protected] Abstract: We report a new approach for electrochemical quantification of single-nucleotide polymorphisms (SNPs) using nanoparticle probes. The principle is based on DNA polymerase I (Klenow fragment)-induced coupling of the nucleotide-modified nanoparticle probe to the mutant sites of duplex DNA under the WatsonCrick base pairing rule. After liquid hybridization events occurred among biotinylated DNA probes, mutant DNA, and complementary DNA, the resulting duplex DNA helixes were captured to the surface of magnetic beads through a biotin-avidin affinity reaction and magnetic separation. A cadmium phosphate-loaded apoferritin nanoparticle probe, which is modified with nucleotides and is complementary to the mutant site, is coupled to the mutant sites of the formed duplex DNA in the presence of DNA polymerase. Subsequent electrochemical stripping analysis of the cadmium component of coupled nanoparticle probes provides a means to quantify the concentration of mutant DNA. The method is sensitive enough to detect 21.5 attomol of mutant DNA, which will enable the quantitative analysis of nucleic acid without polymerase chain reaction preamplification. The approach was challenged with constructed samples containing mutant and complementary DNA. The results indicated that it was possible to accurately determine SNPs with frequencies as low 0.01. The proposed approach has a great potential for realizing an accurate, sensitive, rapid, and low-cost method of SNP detection.
Introduction
Single-nucleotide polymorphisms (SNPs) are point mutations that constitute the most common genetic variation, and they are often closely associated with susceptibility to various common diseases and individual differences in drug metabolism.1,2 Numerous methods and technologies to identify point mutations have been reported to date, including oligonucleotide ligation,3 primer extension,4 allele-specific polymerase chain reaction (PCR),5 microarray,6 TaqManPCR,7 and endonuclease digestion.8 Quantifying SNPs is particularly important for estimating the frequency of SNP alleles in DNA pools.9 To estimate very low SNP frequencies (such as 3. (The noise level is assumed to be the system’s response upon analyzing 0 pM mismatched DNA following the same procedure.) This detection limit corresponds to 21.5 attomole in the 75 µL hybridization solution, which is comparable with that of a bioluminometric assay (14 attomol)24 and a gold nanoparticle-enhanced surface plasmon resonance imaging measurement (1 pM).25 This measurement is more sensitive than that of the enzyme tag (10 pM)14 and colorimetric SNP discrimination (0.4 pmol).26 Also, analyzing the contaminants (such as noncomplementary DNA) using this approach yields a negligible current signal, implying that the contaminants do (24) Zhou, G.; Kamahori, M.; Okano, K.; Chuan, G.; Harada, K.; Kambara, H. Nucleic Acids Res. 2001, 29 (19), e93. (25) Li, Y.; Wark, A.; Lee, H. J.; Corn, R. M. Anal. Chem. 2006, 78, 31583164. (26) Murphy, D.; O’, Brien, P.; Redmond, G. Analyst 2004, 129, 970-974. 10400 J. AM. CHEM. SOC.
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not affect the analysis of the mismatched DNA (results not shown). The results presented in Figure 4 show excellent reproducibility over the entire concentration range. A series of six repetitive measurements of the 1 pM and 25 pM mismatched DNA targets yielded reproducible SWV peaks with a relative standard deviation of 2.9% and 2.3%, respectively (90 min hybridization; not shown). Determination of SNP Frequencies in Constructed DNA Samples. One of the most important applications of the quantification of SNP is to estimate SNP frequency in DNA sample pools. To demonstrate the quantification of SNP frequencies, we used a cytosine-mutated DNA target as a mutant SNP allele and perfect-matched DNA as a wide-type SNP allele to construct an artificial DNA pool. Briefly, mutant DNA and perfect-matched DNA were mixed at different ratios and used as DNA samples. The SNP frequency was calculated with the following equation:
SNP frequency )
I I0 + I100
Here, I is the current intensity produced by the constructed DNA pool sample (containing mutant DNA and perfect-matched DNA), I0 is the current intensity produced by the perfectmatched DNA sample (without mutant DNA), and I100 is the current intensity produced by the mutant DNA sample (without perfect-matched DNA). To start with, samples containing perfect-matched DNA, mutant DNA, and an equal molar mixture of perfect-matched DNA and mutant DNA were analyzed, and results are shown in Figure 5. As expected, a negligible signal was obtained with a perfect-matched DNA sample (0% mutant DNA); the response signal of the equal molar mixture of perfectmatched DNA and mutant DNA is smaller than that of mutant DNA (100%). The results were almost as expected, which
Quantification of Single-Nucleotide Polymorphisms
indicates that the method is applicable for the SNP frequency analysis. A series of artificially prepared DNA samples with known SNP frequencies at 0.01, 0.05, 0.1, and 0.7, were used to challenge the current approach. The observed SNP frequencies in these constructed DNA samples were compared with known values and are listed in Table 1. Each measurement was repeated six times, and the results were highly reproducible. The observed frequencies were consistent with the known values, indicating that the new SNP quantitative approach is reliable. Concluding Remarks
In conclusion, we have designed an electrochemical method based on nanoparticle probes for the specific quantification of single-nucleotide polymorphisms. The principle of the new SNP detection technology is based on DNA polymerase I (Klenow fragment)-induced coupling of the nucleotide-modified nanoparticle probe to the mutant sites of duplex DNA under the Watson-Crick base-pairing rule. Electrochemical stripping analysis is an effective and sensitive electronic transduction method for measuring metallic nanoparticle probes. The method is sensitive enough to detect 21.5 amol mutant DNA, which will enable the quantitative analysis of nucleic acid without PCR
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preamplification. It should be noted, however, that the measuring magnitudes of mutant DNA are controlled by the concentration of DNA probes and the efficiency of hybridization. The approach was challenged with constructed samples containing mutant and complementary DNA. The results indicated that it was possible to very accurately determine SNPs with frequencies as low 0.01. Further experiments that employ genomic DNA samples are under investigation. The proposed approach has a great potential for realizing an accurate, sensitive, rapid, and low-cost method of SNP detection. Acknowledgment. This work is supported by the U.S. Department of Energy’s (DOE’s) Laboratory Directed Research and Development program. The research described in this paper was performed at the Environmental Molecular Sciences Laboratory, a national scientific user facility sponsored by DOE’s Office of Biological and Environmental Research and located at Pacific Northwest National Laboratory, which is operated by Battelle for DOE under contract DE-AC0576RL01830. The author would like to thank Alice Dohnalkova for the TEM experiment. JA070429R
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