(253b) Nanoparticle-Based Assay For DNA Detection And Quantification With Single Nucleotide Discrimination Selectivity

Quantification and selectivity are the other two important aspects , besides, sensitivity, for the evaluation of DNA biosensor devices. DNA quantification is critical for gene expression analysis, detection of DNA mutations or genetic defects, early stage diagnosis of critical illness such as HIV and cancers, and forensic applications. Furthermore, diagnosis of pathogenic and genetic diseases requires the device to have high selectivity that can discriminate single nucleotide mismatches. Single nucleotide polymorphisms (SNPs) are the most abundant form of genetic variation that occur once every 100?300 bases and there are greater than 3 million SNPs in the human genome. Identify these SNPs and associate individual SNPs with specific diseases and pharmacological responses are clinically important for medical diagnostics, disease prevention and prognostics. Currently, real-time polymerase chain reaction (RT-PCR) is one of the most frequently used methods for DNA quantification and SNP discrimination in life science and clinical research. However RT-PCR is a time-consuming and labor intense process, and its selectivity is not always satisfactory even with sophisticated optimizations. Previously, we have shown that gold nanoparticle-DNA (nAu-DNA) conjugates bearing definite number of short DNA (< 20 bases) can be prepared by gel electrophoresis isolation followed by restriction endonuclease manipulation of the nAu-bound DNA. In this study, we described a novel gold nanoparticle (nAu) based assay methodology that has reliable quantification ability and SNP discrimination selectivity. In this assay, two sets of specially designed nAu-DNA conjugates are fabricated via the gel electrophoresis and restriction endonuclease manipulation methods. These two sets of conjugates with definite number (1, 2, 3?) of short single stranded DNA (ssDNA) probes are not directly complementary to each other. After mixing, these conjugates do not recognize and group each other until a target DNA that is complementary to both sets of conjugates is introduced. Only conjugate groupings with defined structure (dimers or trimers) can form due to definite number of DNA strands on each nAu. The resulting conjugate groupings are characterized and quantified by agarose gel electrophoresis. The size differentiation ability of gel electrophoresis allows strict discrimination between different conjugate structures (monomer, dimer and trimer) and enables precise quantification of target DNA samples. Furthermore, a strong discrimination between perfectly matched and single base mismatched DNA is achieved since only the perfectly matched target DNA allows the formation of conjugate groupings. This novel assay methodology is particularly suitable for quantitative DNA analysis and SNP discrimination, and is expected to find application in the diagnosis of genetic & infective disease, forensic analysis as well as biodefense applications.