All existing global gene expression assay techniques require relatively large quantities of analyte;in order to use them with the minute amount of mRNA present in a few or a single cell, enzymatic amplification is required . This process is time consuming, technically difficult, and expensive. More importantly, the amplification process itself biases the sample in a way that prohibits truly quantitative analysis . This, combined with specific limitations of microarrays and DNA sequencing, results in no straightforward method to study transcription in single cells. To solve this problem we aim to directly identify individual gene transcript molecules (in the form of cDNA) via atomic force microscopy.
The Specific Aims of this Application are to combine the building blocks of hardware, software and chemistry that we have developed into an integrated, functional system. The improvement we are proposing will significantly impact medicine by reducing time, cost and technical complexity of small sample transcriptional profiling;and will lower the bioinformatics burden by producing easier to interpret, better quality of information.
Project Narrative The long term goal of our research is to dissect, understand, and control the biology of single cells in complex tissues, such as brain, or in malignant tumors. Furthering this body of work requires that we address an unsolved problem in single cell molecular analysis: the lack of a method to routinely, reliably and inexpensively determine global gene transcriptional activity. The nanotechnologies we are developing will significantly impact medicine by reducing time, cost and technical complexity of small sample transcriptional profiling;and will lower the bioinformatics burden by producing easier to interpret, better quality of information.
|Mikheikin, Andrey; Olsen, Anita; Leslie, Kevin et al. (2017) DNA nanomapping using CRISPR-Cas9 as a programmable nanoparticle. Nat Commun 8:1665|
|Mikheikin, Andrey; Olsen, Anita; Picco, Loren et al. (2016) High-Speed Atomic Force Microscopy Revealing Contamination in DNA Purification Systems. Anal Chem 88:2527-32|
|Kim, Diane N H; Teitell, Michael A; Reed, Jason et al. (2015) Hybrid random walk-linear discriminant analysis method for unwrapping quantitative phase microscopy images of biological samples. J Biomed Opt 20:111211|
|Mikheikin, Andrey; Olsen, Anita; Leslie, Kevin et al. (2014) Atomic force microscopic detection enabling multiplexed low-cycle-number quantitative polymerase chain reaction for biomarker assays. Anal Chem 86:6180-3|
|Zangle, Thomas A; Teitell, Michael A; Reed, Jason (2014) Live cell interferometry quantifies dynamics of biomass partitioning during cytokinesis. PLoS One 9:e115726|
|Zangle, Thomas A; Chun, Jennifer; Zhang, Jin et al. (2013) Quantification of biomass and cell motion in human pluripotent stem cell colonies. Biophys J 105:593-601|
|Chun, Jennifer; Zangle, Thomas A; Kolarova, Theodora et al. (2012) Rapidly quantifying drug sensitivity of dispersed and clumped breast cancer cells by mass profiling. Analyst 137:5495-8|
|Sundstrom, Andrew; Cirrone, Silvio; Paxia, Salvatore et al. (2012) Image analysis and length estimation of biomolecules using AFM. IEEE Trans Inf Technol Biomed 16:1200-7|
|Reed, Jason; Hsueh, Carlin; Lam, Miu-Ling et al. (2012) Identifying individual DNA species in a complex mixture by precisely measuring the spacing between nicking restriction enzymes with atomic force microscope. J R Soc Interface 9:2341-50|
|Hsueh, Carlin; Chen, Haijian; Gimzewski, James K et al. (2010) Localized nanoscopic surface measurements of nickel-modified mica for single-molecule DNA sequence sampling. ACS Appl Mater Interfaces 2:3249-56|