Accurate measurement of nucleic acid concentrations is essential in many areas of genome research and its application to human health. Sensitive, precise and reliable quantitative nucleic acid tests are increasingly necessary in many important public health problems, including cancer, infectious disease and prenatal diagnostics. However, there is currently a lack of practical technologies capable of high precision measurements, creating an unmet need in research and clinical applications where very small differences in concentration must be discerned. We have recently developed a high-sensitivity counting method with the statistical power of digital PCR, yet can be multiplexed to measure many genes simultaneously. In this proposal, we will develop this technology into a single molecule detection system which will allow absolute counting of single copies of nucleic acids. For phase I of this proposal, our objective is to develop a prototype assay kit and detector to make precise measurements of gene expression. Although many techniques in modern molecular biology allow for the relative quantitation of nucleic acids, digital PCR is the only method capable of absolute quantitation. However, it requires expensive reagents and instruments, and is limited to measurements of a single target at a time. In contrast, our novel method first randomly labels every copy of a DNA molecule with an oligonucleotide barcode tag. After PCR amplification, a small detector panel of complementary oligonucleotides is constructed to detect the number of different barcode tags, which reveals the number of original molecules in solution. This transforms the difficult task of counting individual molecules of identical DNAs into the simple process of detecting the number of different amplified barcoding sequence tags present. Because our method expands identical molecules into chemical space, we can perform the measurement in a single tube, making it much simpler and amenable to multiplex target detection than digital PCR where a very large number of separate containers is required. Control nucleic acids of known concentrations will be tested, and independent measurements will be conducted with digital PCR in order to validate the technique. Our company is comprised of an exceptionally strong team of successful innovators and scientists/engineers with significant achievements in both research and product commercialization settings. Additionally, we have established collaborations with scientists at the Stanford University Genome Technology Center, giving us access to instruments and expertise available at this world class research facility.
We propose to develop a highly sensitive system capable of precise measurements of the absolute number of copies of nucleic acid molecules. This simple, yet accurate approach will be able to measure very small changes in nucleic acid concentrations. Applications will include absolute calibrations for sequencing and microarray experiments, single cell RNA and DNA analysis, and will be valuable in many clinical settings where absolute or high-precision assays are required.
|Fu, Glenn K; Wilhelmy, Julie; Stern, David et al. (2014) Digital encoding of cellular mRNAs enabling precise and absolute gene expression measurement by single-molecule counting. Anal Chem 86:2867-70|
|Fu, Glenn K; Xu, Weihong; Wilhelmy, Julie et al. (2014) Molecular indexing enables quantitative targeted RNA sequencing and reveals poor efficiencies in standard library preparations. Proc Natl Acad Sci U S A 111:1891-6|