Colorimetric SNP Genotyping Based on Allele‐Specific PCR by Using a Thiol‐Labeled Primer

Abstract

chemistry. [7] However, these methods usually require multiple manipulations, high levels of technical expertise, and expensive instruments, all of which makes them unsuitable for pointof-care testing (POCT) applications. Consequently, a great incentive exists for the development of simple, rapid, and costeffective SNP genotyping methods that are suitable for POCT in facility-limited environments. Colorimetric methods fulfill most of the above requirements as they enable cost-effective and on-site detection without the need for sophisticated equipment. To date, a number of such methods for SNP detection that utilize, for example, metal nanoparticles, [8] peroxidase-mimicking DNAzymes, [9] and peptide nucleic acids (PNAs), [1] have been reported. Among these, gold nanoparticle (AuNP)-based approaches [10–12] have received great attention owing to their unique optical properties, robustness, and high surface areas, which make them ideally suited as signaling probes in colorimetric-detection platforms. [12] However, the requirement for modification of the AuNP surface with oligonucleotide probes and/or the need for precise temperature control during the assay significantly limits applications of AuNP-based methods. In recent studies, we developed a novel strategy based on unmodified AuNPs for the colorimetric detection of target nucleic acids that are amplified by thiol-labeled primers without time-consuming and complicated procedures. [13] We successfully demonstrated its diagnostic utility by reliably identifying Chlamydia trachomatis infection. Here, we have extended our previous strategy for nucleic acid detection by devising a novel method for SNP identification that incorporates a modified allele-specific PCR (ASPCR) method. In principle, ASPCR is a cost-effective procedure that characterizes SNPs based on differences between the PCR efficiency of allele-specific primers. [14] The primers consist of sequences adjacent to the polymorphic site that are complementary to the allelic variant and differ only in the terminal nucleotide at the 3’-end. [2] In the first step of our new assay procedure, ASPCR is performed with a thiolated primer. In the second step, AuNPs are mixed with the products and then subjected to salt-induced aggregation. The resulting color of the solution (i.e., red or blue) indicates the genotype at the SNP site because only the thiolated PCR products provide the AuNPs with a significant resistance to salt-induced aggregation and inhibit the red-to-blue color transition. The strategy we have devised for SNP identification is schematically illustrated in Figure 1 for the detection of a single base mutation. In the first step, ASPCR is performed separately in four different reaction tubes containing each of the four thiolated forward primers, which differ only in the terminal base at their 3’-ends (A, T, C and G). When the base at the 3’end of the forward primer is complementary to the antisense strand of the genomic target DNA, PCR amplification occurs and generates PCR amplicons labeled with a thiol group at the 5’-end of one strand. In contrast, the other three primers, which have a noncomplementary nucleotide at their 3’-ends, cannot be extended and thus they fail to generate PCR amplicons. The existence of the thiol-labeled PCR products is then colorimetrically determined by sequentially adding unmodified AuNPs and NaCl to each tube. When thiolated PCR amplicons are mixed with AuNPs, they bind to the surface of unmodified AuNPs through a strong gold–thiol interaction. The electrostatic repulsion between the AuNPs is enhanced as a consequence of the large number of negative charges on their surfaces due to the negatively charged phosphate backbone of the bound DNA. In addition, steric repulsion between the AuNPs is increased because the longer (compared to the diameter of AuNPs) DNA grafted onto the AuNPs surface forms a thick polymeric barrier that prevents the particles from approaching each other. [15] As a consequence of these “electrostatic/steric stabilization effects”, [16] salt-induced aggregation of AuNPs is significantly inhibited, and the solution remains red when salt is added. In contrast, in the absence of the thiol-labeled PCR amplicon, AuNPs undergo immediate aggregation upon salt addition; this results in a colorimetric transition from red to blue. By employing this strategy, the genotypes at mutation sites can be conveniently determined based on the color of the sample. To prove the conceptual basis of this novel strategy for SNP genotyping and to confirm the crucial role played by thiolated primers in the AuNPs-based colorimetric assay, ASPCR was per