Pulsed-multiline excitation for color-blind fluorescence detection
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Abstract
The present invention provides a technology called Pulse-Multiline Excitation or PME. This technology provides a novel approach to fluorescence detection with application for high-throughput identification of informative SNPs, which could lead to more accurate diagnosis of inherited disease, better prognosis of risk susceptibilities, or identification of sporadic mutations. The PME technology has two main advantages that significantly increase fluorescence sensitivity: (1) optimal excitation of all fluorophores in the genomic assay and (2) “color-blind” detection, which collects considerably more light than standard wavelength resolved detection. This technology differs significantly from the current state-of-the-art DNA sequencing instrumentation, which features single source excitation and color dispersion for DNA sequence identification. Successful implementation of the PME technology will have broad application for routine usage in clinical diagnostics, forensics, and general sequencing methodologies and will have the capability, flexibility, and portability of targeted sequence variation assays for a large majority of the population.
43 Citations
84 Claims
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1-38. -38. (canceled)
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39. A method of identifying sample components comprising:
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(a) preparing a sample comprising sample components, a first dye and a second dye;
(b) placing the sample in the beam path of a first excitation line and a second excitation line;
(c) sequentially firing the first excitation line and the second excitation line;
(d) collecting fluorescence signals from the samples as a function of time; and
(e) sorting the fluorescence by each excitation line'"'"'s on-time window, wherein the sample components are identified. - View Dependent Claims (40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60)
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61. A method of identifying sample components comprising:
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(a) obtaining a biological sample;
(b) labeling said sample with one or more fluorophores;
(c) separating components of said sample; and
(d) detecting said sample components with a device wherein said device comprises;
one or more lasers configured to emit two or more excitation lines, each excitation line having a different excitation wavelength;
a timing circuit coupled to the one or more lasers and configured to fire the two or more excitation lines sequentially according to a timing program to produce time-correlated fluorescence emission signals from the sample; and
a non-dispersive detector positioned to collect the time-correlated fluorescence emission signals;
wherein said detector collects time correlated data from said sample comprising fluorescent emissions of the sample as a result of irradiation by the one or more excitation lines. - View Dependent Claims (62, 63, 64, 65, 66, 67, 68, 69, 70, 71)
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72. A device comprising:
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(a) one or more lasers having two or more excitation lines;
(b) one or more beam steering mirrors wherein said excitation lines each strike said mirrors;
(c) a first prism, wherein said two or more excitation lines strike one surface and exit from a second surface of said first prism; and
(d) a second prism at an angle relative to said first prism, wherein said two or more excitation lines strike one surface of said second prism after exiting said first prism and exit said second prism, wherein said two or more excitation lines are substantially colinear or coaxial after exiting said second prism. - View Dependent Claims (73, 74)
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75. A method of illuminating a sample comprising:
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(a) steering two or more excitation lines onto a first surface of a first prism;
(b) steering two or more excitation lines from the second surface of said first prism to a first surface of a second prism;
wherein said second prism is angled about 45°
from said first prism;
(c) steering said two or more excitation lines onto a sample after exiting second surface of said second prism, wherein said two or more excitation lines are substantially colinear or coaxial after exiting said second prism. - View Dependent Claims (76, 77)
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78. A method of controlling a sequence of excitation lines comprising:
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obtaining a TTL circuit comprising an electronic stepper wherein said circuit is operationally connected to one or more lasers having two or more excitation lines; and
controlling the sequential firing of the one or more lasers having two or more excitation lines with a clock pulse from the circuit, wherein the frequency of firing one laser is equivalent to the frequency of firing a second laser, but phased shifted so that one or more lasers having two or more excitation lines can be sequentially pulsed. - View Dependent Claims (79, 80, 81, 82)
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83. A method of controlling a sequence of excitation lines comprising:
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obtaining a TTL circuit comprising an electronic stepper wherein said circuit is operationally connected to two or more lasers; and
controlling the sequential firing of the two or more lasers with a clock pulse from the circuit, wherein the frequency of firing a first laser is different from the frequency of firing a second laser. - View Dependent Claims (84)
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Specification