Ultrasensitive micro rna quantification
AssigneeThe Board of Trustees of the University of Illinois
InventorsAndrew SMITH, Lucas David SMITH
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Abstract
The present invention relates to an ultrasensitive assay platform for the detection of nucleic acids such as microRNAs (miRNAs), which are important biomarker for diseases including cancer. The platform allows high throughput detection of multiple nucleic acid sequences miRNAs on the single-molecule level using fluorescence labeling, molecular barcoding, and flow based detection and multiparametric data analysis.
Claims32
A system for quantifying nucleic acid at a single molecule level in a biological sample, the system comprising:
- (1) a sub-system for extending the nucleic acid to form an amplicon,
- (2) a subsystem for labeling the amplicon with more than one fluorescent dye to form a labeled nucleic acid amplicon, wherein the labeled nucleic acid amplicon is labeled by:
- (i) hybridization of the nucleic acid amplicon with a fluorescent dye conjugated to a single-stranded nucleic acid or nucleic acid analog,
- (ii) binding or intercalation of single-stranded nucleic acid-specific or double-stranded nucleic acid-specific fluorescent dyes with the amplicon, and/or
- (iii) addition of the fluorescent dye into the nucleic acid amplicon by enzymatic incorporation of a fluorescent dye labeled nucleoside triphosphate or deoxyribonucleoside 5′-triphosphate,
- (3) a fluorescence detection sub-system comprising an excitation source and fluorescence detector for excitation of the fluorescently labeled amplicon and measuring fluorescence emission from individual molecules of the fluorescently labeled amplicon, wherein the excitation source comprises a laser source, an incandescent light source, or an light emitting light source.
- (4) software to record and analyze the fluorescence emission from the detection subsystem.
A method for quantifying nucleic acid at a single molecule level in a biological sample, comprising:
- 1) extending the nucleic acid and labelling a resulting amplicon with more than one fluorescent dye to form a labeled amplicon, by
- (i) hybridizing of the nucleic acid amplicon with a fluorescent dye conjugated to a single-stranded nucleic acid or nucleic acid analog,
- (ii) binding or intercalation of single-stranded nucleic acid-specific or double-stranded nucleic acid-specific fluorescent dyes with the amplicon, and/or
- (iii) addition of the fluorescent dye into the nucleic acid amplicon by enzymatic incorporation of a fluorescent dye labeled nucleoside triphosphate or deoxyribonucleoside 5′-triphosphate,
- 2) exciting the fluorescently labeled amplicon by an excitation source comprising a laser, an incandescent light, or an LED, and measuring the fluorescence emission from individual molecules of the fluorescently labeled amplicon,
- 3) recording and analyzing data of the measured fluorescence emissions.
Dependencies are inferred from the claim text.