Sample preparation of nucleic acids is essential for all genomic analyses, and yet it is the primary source of variability and requires tedious manual labor. For samples with relatively low abundance (<100 ng or roughly 20,000 mammalian cells or less), current sample preparation methods also result in crippling material loss and low repeatability. For small RNA, current technologies can result in severe sequence or length biases. We here propose a novel method and product which achieves nucleic acid extraction, purification, and quantitation from complex samples using isotachophoresis (ITP). ITP is an electrophoresis method which can be used to highly selectively extract target nucleic acids from complex samples including blood, urine, and cell culture. Proposed is the development of novel, low-cost plastic chips and a benchtop reader and control unit for automated extraction of DNA or RNA from complex lysate samples. The method offers the unique capability of processing low abundance samples: Providing high efficiency extraction and purification from samples containing anywhere from 1 microgram down to 1 picogram of nucleic acid (1000x lower than competing technologies). The entire process is achieved with computer-controlled electrodes, is fully automated, and will be performed in less than 30 min. The electric fields of ITP also provide strong focusing of the purified nucleic acid and the system will use this to enable quantitation with 100x better sensitivity than competing technologies. The extracted nucleic acid will be highly pure and immediately compatible with a wide range of downstream analyses including microarrays, enzymatic amplification methods (including PCR), and sequencing. In this Phase I, the effort targets extraction from 1 to 60 microliter sample volumes in a system capable of processing four samples in parallel. Phase II will center around the development of a monolithic chip capable of eight samples each with 1 to 100 microliter volumes. The proposed prototypes and demonstrations will represent a major development in the science of sample preparation and a significant step in our objective to commercialize and sell this unique life sciences tool for both research and clinical applications.
The science of genomics aims to revolutionize medicine and biology, but still relies on 20+ year old sample preparation methods which require tedious manual labor, are error prone, and create biases in downstream analyses. Proposed is a method and benchtop tool with potential to revolutionize sample preparation across all of genomics: Isotachophoresis (ITP) based extraction, purification, and quantitation of nucleic acids from raw samples. The process is fully automated, fast, and provides unprecedented access to extremely low abundance (low DNA content) samples. ITP can also quantify extracted nucleic acids with unprecedented sensitivity. The proposed project will demonstrate low-cost plastic chips and a benchtop tool which provides high efficiency sample preparation for virtually all genomic research and clinical applications.
