Closed-Tube PCR with Nested Serial Invasion Probe Visualization Using Gold Nanoparticles

Abstract

Abstract BACKGROUND Detecting DNA biomarkers related to personalized medicine could improve the outcome of drug therapy. However, personalized medicine in a resource-restrained hospital is very difficult because DNA biomarker detection should be performed by well-trained staff and requires expensive laboratory facilities. METHODS We developed a gold nanoparticle–based “Tube-Lab” to enable DNA analysis in a closed tube. Gold nanoparticle–modified probes (GNPs) were used to construct an inexpensive and simple DNA sensor for signal readout. The method consists of 3 steps (template amplification, sequence identification, and GNP-based signal readout), bridged by an invasive reaction. With temperature control at each step, the 3 reactions proceed sequentially and automatically in a closed tube without any liquid transfer. We used Tube-Lab to detect different biomarkers in blood, tissue, and plasma, including US Food and Drug Administration–approved pharmacogenomic biomarkers (single nucleotide polymorphisms, somatic mutations). RESULTS The combination of PCR-based template replication and invader-based signal amplification allowed detection of approximately 6 copies of input DNA and the selective pick up 0.1% mutants from large amounts of background DNA. This method highly discriminated polymorphisms and somatic mutations from clinical samples and allowed a “liquid biopsy” assay with the naked eye. CONCLUSIONS Tube-Lab provides a promising and cost-effective approach for DNA biomarker analysis, including polymorphisms and somatic mutations from blood DNA, tissue DNA, or circulating tumor DNA in plasma, which are critical for personalized medicine.