The most common method employed for carrying out quantitative PCR (qPCR) is via real-time PCR (RT-PCR), which is widely employed in both clinical diagnostics and in basic biomedical studies. In RT- PCR, the presence and concentration of the target DNA is detected and quantified in real time, most frequently by using a fluorescent reporter probe. Despite the power of RT-PCR in identifying and quantifying target DNA from complex samples, it suffers from the inability to quantify low concentrations of DNAs. Yet, the ability to quantify low-copy-number of DNAs present in the sample is becoming increasingly important, such as in the detection of pathogens and in diagnostics. To overcome this issue, the notion of digital PCR (dPCR) has been put forth more than a decade ago. In dPCR, the sample is divided into an array of small volumes such that each volume contains only one copy of the DNA molecule in the sample while the majority of the volumes contain no DNA molecules. PCR amplification of the array of volumes will result in fluorescence only in the few small volumes that each contains a single copy of DNA. The DNA copy number is easily and accurately determined by simply counting the number of small volumes that contain a copy of DNA (i.e. fluorescent). Despite the conceptual appeal of dPCR, its implementation can be challenging. Our proposal aims to address this challenge. We have recently discovered a simple and robust method for generating a large array (thousands) of small volumes spontaneously. We believe this method will greatly simplify the fluidics associated with digital PCR. Furthermore, by increasing the dynamic range of dPCR, we plan to develop a next-generation platform for carrying out qPCR with enhanced sensitivity and accuracy, and at a reduced cost.
Our specific aims are: (1) Develop and demonstrate digital PCR using our fluidic self-digitization chip. (2) Increase the dynamic range and throughput of our digital PCR platform. (3) Demonstrate our digitization method can be employed to make quantitative other non-PCR nucleic acid amplification schemes (e.g. NASBA and LAMP). This RFA stated that """"""""Projects should propose tools that can be used by a wide range of biomedical or clinical researchers"""""""". We believe our proposed project addresses this stated goal, because we aim to develop the next generation of quantitative PCR instruments that offers greatly improved sensitivity and accuracy, while at the same time reduces the cost of the instrument and assay.
(provided by applicant): Polymerase Chain Reaction (PCR) is a ubiquitous tool used in biology and medicine. Our project aims to develop the next generation of quantitative PCR instruments that offers greatly improved sensitivity and accuracy, while at the same time reduces the cost of the instrument and the assay. Successful development of the proposed project will result in a tool that will be used by a wide range of biomedical and clinical researchers.
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|Gansen, Alexander; Herrick, Alison M; Dimov, Ivan K et al. (2012) Digital LAMP in a sample self-digitization (SD) chip. Lab Chip 12:2247-54|