Available Technology

METHOD FOR MEASURING THE SIZE OF SINGLE NUCLEIC-ACID MOLECULES

• Providing a sample that includes nucleic-acid molecules of unknown size and concentration• Labeling the sample with a fluorescent dye such that the amount of dye labeling each nucleic-acid molecule is reliably proportional to the size of the moleculeo Measuring the unknown concentration of nucleic acid in the sampleo Passivating the surface of the reactor that will be used for labeling t1cie sample o Incubating the sample with fluorescent dye for an amount of time and at atemrerature that results i n homogeneous labeling of all nucleic-acid molecules inthe sample• Dispersing the sample in a suspension of fluorescent nanoparticles that have a negative surface charge and a distinct fluorescence spectrum from the dye-labeled nucleic-acid molecules• Placing the nucleic-acid molecules onto a charge-selective surface for wide-field imaging o A coverglass substrate with a high surface density of positive charge toselectively attract and bind the nucleic-acid molecules and nanoparticles and repel free dye molecules into solution• Orienting and positioning the imaging surface within the depth of field of the imaging system without perturbing the dye molecules labeling the nucleic-acidso Using a substrate holder that enables tip and tilt adjustment of the imaging surface with respect to the focal plane of the objective lenso Positioning the imaging surface within the focal plane of the objective lens using the fluorescence signal from the nanoparticles as feedback• Using a light-emitting diode to supply exciting radiation to many nanoparticles simultaneously over a wide field• Irradiating the sample to excite the fluorescent dye labeling the nucleic-acid moleculeso Using a light-emitting diode to supply exciting radiation to many single nucleic­ acid molecules simultaneously over a wide field• Detecting the radiation emitted by many single nucleic-acid molecules i n the sample with a wide-field imaging sensoro Using a complimentary-metal-oxide-semiconductor  imaging sensor to detect the radiationMoving the sample holder to detect emitted radiation from additional regions of the imaging surface that have not been exposed to exciting radiation• Determining the size of single nucleic-acid molecules in the sample based on a quantitative calibration between the intensity of detected radiation in arbitrary units and nucleic acid size in bases.o Image correction to account for non-uniformity  in the imaging sensor and the spatial distribution of the exciting radiation from the light-emitting diodeo Measuring the intensity of detected radiation from single nucleic-acid molecules in the acquired imageso Establishing a calibration between the intensity of detected radiation and nucleic­ acid molecule size using pure samples of single known sizes of nucleic-acid moleculeso Fitting an appropriate function to model the dependence of the coefficient of variation of the intensity of emitted radiation on nucleic-acid molecule sizeo Taking as an input to this model the measured size of single nucleic-acid molecules in the sample of nucleic-acid molecules of unknown sizeo Taking as the uncertainty on the measured size of single nucleic-acid molecules the output of the model functionMeasuring the size of nucleic-acid molecules is important in a variety of applications ra nging from criminal forensics to clinical diagnostics. The invention disclosed herein advances conventional methods and instruments used to size single nucleic-acid molecules and provides an unprecedented combination of limit of detection, throughput,  accuracy, precision, and repeatability.   The methods and apparatus can be used in clinical diagnostics. A more detailed discussion of the invention is provided in the Detailed Description.

Inventors: 
Copeland, Craig Robert

Stavis, Samuel M.

Patent Number: 
10,379,038
Internal Laboratory Ref #: 
16-044
Patent Issue Date: 
August 13, 2019
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