The long term objective of this proposal is to increase the sensitivity of DNA concentration measurements by 1000X over the state of the art. In addition, the microfluidic platform we will develop to accomplish this will also enable, for the fist time, the targeted recovery of genome-size DNA fragments out of a heterogeneous sample. Nucleic acid analysis through technologies like microarrays and next-generation sequencing are transforming the way that many biological questions are addressed. The power of these approaches stems from their ability to obtain system-wide information with single molecule detail. However, a major technical barrier that often prevents their effective application is that many biological systems are too complex to be straightforwardly sequenced: Even with bioinformatic algorithms, the billions of short, sequence dreads are often too complex to piece together into useful information. To address this challenge, we will develop a technology that allows nucleic acids to be quantitated and sorted in a heterogeneous sample. Importantly, this method will utilize specific multiplexedTaqMan PCR assays performed in giant-unilamellarvesicles (GUVs);this will make it much more specific and targetable than methods that sort DNA based on size or staining properties. Moreover, by assaying individual molecules or cells in GUVs, we will be able to perform billions of PCR assays in parallel, enabling massive, heterogeneous samples to be screened to identify and recover extremely rare targets. This basic technology will be broadly useful throughout biological research and has immediate human health impacts, including for detecting and sequencing cancer DNA in the blood early in the disease, analyzing genetic heterogeneity in tumor cells, and identifying immune cells latently infected with HIV.
The aims are:
Specific Aim 1 : Demonstrate microfluidic generation and FACS sorting of thermostable femtoliterGiant-Unilamellar Vesicles. We will develop the microfluidic hardware and processes for generating billions of monodisperse femtoliter GUVs. We will also optimize processes to FACS GUVs, both into """"""""positive"""""""" and """"""""negative"""""""" pools and, individually, into wells on a microliter plate.
Specific Aim 2 : Optimize and characterize GUV-PCR and benchmark against aqueous droplets. We will explore different PCR reagents to optimize the GUV-PCRs and benchmark the efficiency of these reactions against ones performed in aqueous-in-oil droplets generated with our own microfluidics and with the Bio-rad QX100 digital PCR machine. We will measure efficiency using endpoint fluorescence, fraction of positive reactors, and yield of DNA recovered out of the reactors. We will also optimize protocols for rupturing GUVs to access their contents.
Specific Aim 3 : Demonstrate 1000X greater sensitivity than competing platforms by performing over 1 billion digital GUV-PCRs, and recovery of positive molecules with FACS. We will demonstrate the superiority of GUVs for digital PCR by performing 1000X more reactions than is possible with existing droplet technologies. We will also demonstrate the ability to recover rare DNA molecules by FACS sorting the positive GUVs.
The proposed study will develop a system for analyzing and sorting DNA for important biological and industrial applications through the development of powerful microfluidic technologies. The technologies developed will advance the fields of genomics and single cell analysis and will provide new tools to understand human disease. These technologies should ultimately have broad biomedical, biotechnological, and industrial applications.
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