title: Real-time analysis and selection of methylated DNA by fluorescence-activated single molecule sorting in a nanofluidic channel.
abstract: Epigenetic modifications, such as DNA and histone methylation, are responsible for regulatory pathways that affect disease. Current epigenetic analyses use bisulfite conversion to identify DNA methylation and chromatin immunoprecipitation to collect molecules bearing a specific histone modification. In this work, we present a proof-of-principle demonstration for a new method using a nanofluidic device that combines real-time detection and automated sorting of individual molecules based on their epigenetic state. This device evaluates the fluorescence from labeled epigenetic modifications to actuate sorting. This technology has demonstrated up to 98% accuracy in molecule sorting and has achieved postsorting sample recovery on femtogram quantities of genetic material. We have applied it to sort methylated DNA molecules using simultaneous, multicolor fluorescence to identify methyl binding domain protein-1 (MBD1) bound to full-duplex DNA. The functionality enabled by this nanofluidic platform now provides a workflow for color-multiplexed detection, sorting, and recovery of single molecules toward subsequent DNA sequencing.
authors.affiliation: Department of Electrical and Computer Engineering, Cornell University, Ithaca, NY 14853, USA.
authors.forename: Benjamin R
authors.initials: BR
authors.lastname: Cipriany
authors.forename: Patrick J
authors.initials: PJ
authors.lastname: Murphy
authors.forename: James A
authors.initials: JA
authors.lastname: Hagarman
authors.forename: Aline
authors.initials: A
authors.lastname: Cerf
authors.forename: David
authors.initials: D
authors.lastname: Latulippe
authors.forename: Stephen L
authors.initials: SL
authors.lastname: Levy
authors.forename: Jaime J
authors.initials: JJ
authors.lastname: Benítez
authors.forename: Christine P
authors.initials: CP
authors.lastname: Tan
authors.forename: Juraj
authors.initials: J
authors.lastname: Topolancik
authors.forename: Paul D
authors.initials: PD
authors.lastname: Soloway
authors.forename: Harold G
authors.initials: HG
authors.lastname: Craighead
mesh_terms: D004247:DNA / Q000032:analysis / Q000235:genetics* / Q000378:metabolism; D019175:DNA Methylation; D004268:DNA-Binding Proteins / Q000378:metabolism; D005453:Fluorescence; D006801:Humans; D046210:Microfluidic Analytical Techniques / Q000295:instrumentation / Q000379:methods*; D018613:Microscopy, Confocal; D036103:Nanotechnology / Q000295:instrumentation / Q000379:methods*; D011485:Protein Binding; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods; D015203:Reproducibility of Results; D013997:Time Factors; D014157:Transcription Factors / Q000378:metabolism
chemical_list: D004268:DNA-Binding Proteins; C066141:MBD1 protein, human; D014157:Transcription Factors; D004247:DNA