The goal of this project is to develop a micro- and nanofluidic platform for the rapid, low-cost identification of cell lines that can be used directly in laboratories conducting cell line research. A cell suspension is introduced to a device where automated cell lysis, DNA extraction, and DNA cutting with a rare-cutting restriction endonuclease are performed. The resulting primary restriction fragments are then mapped using our high-resolution DNA mapping technology, which uses a second restriction endonuclease to generate an ordered map of smaller fragments that can be sized at a resolution of 100 bp. The contours of these individual maps, each one originating from a fragment generated by the first digestion, are sensitive to structural variants in the genome. This approach is called RFLP2 to signify the greater information content engendered by sequential, rather than parallel, dual enzymatic fragmentation. Cell lines will be differentiated from each other and from inter-species contaminants based on a unique fingerprint generated from tens to hundreds of these single-molecule maps. In this Phase I project, in-silico experiments will be used to determine the optimal enzyme combinations to use for the primary and secondary digestions to generate the greatest number of highly identifying maps? with a full consideration of real-world measurement errors. DNA extraction and digestion with the primary restriction endonuclease will also be optimized for subsequent integration with the secondary digestion and high-resolution mapping. We believe that the platform?s low cost and ease of use, coupled with researchers? ability to qualify cell lines at multiple checkpoints within their own laboratories, will encourage routine cell line identification, which is critically needed to improve the quality of and confidence in important basic and preclinical research.
Project Terms: Adoption; base; Basic Science; Biomedical Research; Cell Count; Cell Extracts; Cell Line; Cells; Clinical; Computer Simulation; cost; cost effective; Coupled; Cytolysis; Databases; Devices; Digestion; DNA; DNA mapping; DNA Restriction Enzymes; endonuclease; Ensure; Enzymes; experimental study; Fingerprint; flexibility; Fluorescence; Funding; Genome; Genomic DNA; Goals; Hour; improved; Individual; Laboratories; Lasers; Lead; Length; Liquid substance; mammalian genome; Maps; Measurement; Measures; Methods; nanochannel; nanofluidic; new technology; North Carolina; Peptide Sequence Determination; Phase; Population; Positioning Attribute; pre-clinical research; preclinical study; Probability; Process Measure; Reader; Reagent; Reproducibility of Results; Research; Research Personnel; research study; Resolution; restriction enzyme; Restriction Mapping; Sampling; single molecule; Site; Stains; Standardization; success; Surveys; Suspensions; tandem mass spectrometry; Techniques; Technology; Testing; Time; TimeLine; Universities; Variant; wasting; whole genome; Work;
