A Rapid, Low-Cost Application of High-Resolution DNA Mapping for Cell Line Identification

The goal of this project is to develop a microand nanofluidic platform for the rapidlow cost identification of cell lines that can be used directly in laboratories conducting cell line researchA cell suspension is introduced to a device where automated cell lysisDNA extractionand DNA cutting with a rare cutting restriction endonuclease are performedThe resulting primary restriction fragments are then mapped using our high resolution DNA mapping technologywhich uses a second restriction endonuclease to generate an ordered map of smaller fragments that can be sized at a resolution ofbpThe contours of these individual mapseach one originating from a fragment generated by the first digestionare sensitive to structural variants in the genomeThis approach is called RFLPto signify the greater information content engendered by sequentialrather than paralleldual enzymatic fragmentationCell 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 mapsIn this Phase I projectin 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 mapswith a full consideration of real world measurement errorsDNA extraction and digestion with the primary restriction endonuclease will also be optimized for subsequent integration with the secondary digestion and high resolution mappingWe believe that the platform s low cost and ease of usecoupled with researchersability to qualify cell lines at multiple checkpoints within their own laboratorieswill encourage routine cell line identificationwhich is critically needed to improve the quality of and confidence in important basic and preclinical research A significant percentage of cell lines currently used in scientific research are misidentifiedwhich can lead to misleading and irreproducible results in basic and preclinical studieswasting research funds and increasing the timeline to clinical impactWe propose a low costrapid cell line identification platform based on high resolution restriction mapping at various genome lociutilizing the variation in mammalian genomes to generate fingerprints that can distinguish between cell lines and identify low levels of contaminationWe believe that the platform s low cost and ease of usecoupled with researchersability to qualify cell lines at multiple checkpoints within their own laboratorieswill encourage routine cell line authentication