IPC분류정보
국가/구분 |
United States(US) Patent
공개
|
국제특허분류(IPC7판) |
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출원번호 |
US-0492698
(2012-06-08)
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공개번호 |
US-0178378
(2013-07-11)
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발명자
/ 주소 |
- HATCH, Andrew C.
- LEE, Abraham
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출원인 / 주소 |
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인용정보 |
피인용 횟수 :
0 인용 특허 :
0 |
초록
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Embodiments of the claimed subject matter are directed to the ability to further multiplex in the digital regime using combinatorial color, temporal, and intensity encoding of probe sequences for a greater number of total signal readouts. A digital PCR solution is provide which enables the unique ab
Embodiments of the claimed subject matter are directed to the ability to further multiplex in the digital regime using combinatorial color, temporal, and intensity encoding of probe sequences for a greater number of total signal readouts. A digital PCR solution is provide which enables the unique ability to identify a greater number of fluorescent probe sequences by using multiple color, temporal, and intensity combinations to encode each unique probe sequence. Furthermore, less expensive real-time PCR amplification indicators such as PicoGreen can be used to achieve multiplexed digital PCR based on temporal cues, intensity cues, or intensity and temporal cues combined, thus distinguishing primer pairs at greater degrees with significant cost reductions. These can also be used to enhance controls and normalize results for greater accuracy.
대표청구항
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1. A method for amplifying a DNA sequence with a polymerase chain reaction (PCR), the method comprising: discretizing a volume of concentration comprising a plurality of DNA samples into a plurality of reactor volumes such that a presence of a DNA sample in a reactor volume is digitized;performing a
1. A method for amplifying a DNA sequence with a polymerase chain reaction (PCR), the method comprising: discretizing a volume of concentration comprising a plurality of DNA samples into a plurality of reactor volumes such that a presence of a DNA sample in a reactor volume is digitized;performing a multiplex polymerase chain reaction (PCR) process in the plurality of reactor volumes to amplify a plurality of DNA sequences in each reactor volume;emitting fluorescences in a plurality of spectral wavelengths when an DNA sequence of the plurality of DNA sequences is amplified; andidentifying the DNA sequences amplified according to the emitted fluorescences,wherein the volume of concentration comprises a plurality of primers, each of the plurality of primers corresponding to a DNA sequence of the plurality of DNA sequences and comprising a fluorescent reporter configured to emit fluorescence in a spectral wavelength of the plurality of spectral wavelengths when the corresponding DNA sequence is amplified. 2. The method according to claim 2, wherein emitting fluorescence in a plurality of spectral wavelengths when a DNA sequence is amplified comprises emitting fluorescences from the fluorescent reporters comprised in the plurality of primers in a color combination of a plurality of color combinations, the color combination comprising a combination of spectral wavelengths of the plurality of spectral wavelengths. 3. The method according to claim 2, wherein the color combination corresponds uniquely to a particular DNA sequence of the plurality of DNA sequences. 4. The method according to claim 3, wherein identifying the DNA sequences amplified according to the emitted fluorescences comprises: determining the color combinations comprising the emitted fluorescences; andidentifying the particular DNA sequences corresponding to the emitted color combinations. 5. The method according to claim 1, wherein each fluorescent reporter of the plurality of fluorescent reporters is further configured to emit fluorescence in a spectral wavelength at a pre-defined intensity. 6. The method according to claim 5, wherein the spectral wavelength and the pre-defined intensity uniquely correspond to the DNA sequence. 7. The method according to claim 6, wherein emitting fluorescences in a plurality of spectral wavelengths when a DNA sequence is amplified comprises emitting fluorescence in at least one of: 1) a different spectral wavelength; and 2) at a different pre-defined intensity, for each of the plurality of DNA sequences. 8. The method according to claim 5, wherein the pre-defined intensity in the fluorescence in a spectral wavelength emitted by the fluorescent reporter corresponds to a concentration of the fluorescent reporter comprised in the primer. 9. The method according to claim 5, wherein identifying the DNA sequences amplified according to the emitted fluorescences comprises: determining the intensities and spectral wavelengths comprising the emitted fluorescences; andidentifying the particular DNA sequences corresponding to the emitted intensities and spectral wavelengths. 10. The method according to claim 1, wherein emitting fluorescence in a plurality of spectral wavelengths when a DNA sequence is amplified comprises emitting fluorescences from the fluorescent reporters comprised in the plurality of primers in a color combination of a plurality of color combinations at an intensity of a plurality of pre-defined intensities. 11. The method according to claim 10, wherein a color combination and pre-defined intensity corresponds uniquely to a particular DNA sequence. 12. The method according to claim 11, wherein identifying the DNA sequences amplified according to the emitted fluorescences comprises: determining the color combinations and intensities comprising the emitted fluorescences; andidentifying the particular DNA sequences corresponding to the color combinations and intensities. 13. The method according to claim 1, wherein performing a multiplex polymerase chain reaction (PCR) process in the plurality of reactor volumes to amplify a plurality of DNA sequences in each reactor volume comprises performing a plurality of thermocycles. 14. The method according to claim 13, wherein each fluorescent reporter of the plurality of fluorescent reporters is further configured to emit fluorescence in a spectral wavelength after a pre-defined number of thermocycles have elapsed. 15. The method according to claim 14, wherein the pre-defined number of thermocycles after which a fluorescent reporter emits fluorescence corresponds uniquely to a particular DNA sequence of the plurality of DNA sequences. 16. The method according to claim 15, wherein the volume of concentration further comprises a second plurality of primers corresponding to a plurality of DNA sequences, the second plurality of primers not comprising fluorescent reporters. 17. The method according to claim 16, wherein the pre-defined number of thermocycles after which a fluorescent reporter emits fluorescence for a particular DNA sequence corresponds to the ratio of primers of the plurality of primers to primers of the second plurality of primers. 18. The method according to claim 14, wherein the plurality of primers comprise primers configured bind to DNA sequences at varying efficiencies. 19. The method according to claim 14, wherein a primer of the plurality of primers configured to bind to a DNA sequence with a higher efficiency will emit fluorescence after a lower number of thermocycles has elapsed than a primer configured to bind to a DNA sequence at a lower efficiency. 20. The method according to claim 14, wherein the plurality of primers comprise primers that are configured to bind to a DNA sequence at varying temperatures in the plurality of reactor volumes. 21. The method according to claim 20, wherein the temperature in the plurality of reactor volumes is cycled through a pre-defined range during a thermocycle of the plurality of thermocycles. 22. The method according to claim 14, wherein identifying the DNA sequences amplified according to the emitted fluorescences comprises: determining the numbers of thermocycles performed by the multiplex PCR process prior to emitting fluorescences; andidentifying the particular DNA sequences corresponding to the numbers of thermocycles. 23. The method according to claim 1, wherein the fluorescent reporter comprises an intercalating dye. 24. The method according to claim 1, wherein the fluorescent reporter comprises a probe comprising a fluorophore and a quencher. 25. The method according to claim 24, wherein the problem comprises a TaqMan probe. 26. A system for performing multiplex polymerase chain reaction (PCR), the system comprising: a plurality of DNA samples;a volume of concentration comprising the plurality of DNA samples;a plurality of reactor volumes comprising a discretized distribution of the volume of concentration, each reactor volume being configured such that a presence of a DNA sample is digitized; anda thermal cycler coupled to the plurality of reactor volumes and operable to configure a temperature in the plurality of reactor volumes,wherein the volume of concentration comprises a plurality of primers, each of the plurality of primers is configured to bind to, and amplify a DNA sequence of the plurality of DNA sequences in conjunction with a DNA template,wherein the plurality of primers comprises a plurality of fluorescent reporters configured to emit fluorescence in a plurality of spectral wavelengths when a DNA sequence corresponding to a primer of the plurality of primers is amplified. 27. The system according to claim 26, further comprising a filter for detecting fluorescence emitted by a fluorescent reporter of the plurality of fluorescent reporters. 28. The system according to claim 26, wherein the plurality of fluorescent reporters comprised in the plurality of primers are configured to emit fluorescence in a color combination of a plurality of color combinations, the color combination comprising a combination of spectral wavelengths of the plurality of spectral wavelengths. 29. The system according to claim 28, wherein the color combination corresponds uniquely to a particular DNA sequence of the plurality of DNA sequences. 30. The system according to claim 29, wherein the DNA sequences amplified during a PCR process are identified based on the emitted color combinations. 31. The system according to claim 26, wherein each fluorescent reporter of the plurality of fluorescent reporters is further configured to emit fluorescence in a spectral wavelength at a pre-defined intensity. 32. The system according to claim 31, wherein the spectral wavelength and the pre-defined intensity uniquely correspond to the DNA sequence. 33. The system according to claim 32, wherein the plurality of fluorescent reporters is configured to emit fluorescence in at least one of: 1) a different spectral wavelength; and 2) at a different pre-defined intensity, for each of the plurality of DNA sequences. 34. The system according to claim 31, wherein the pre-defined intensity in the fluorescence in a spectral wavelength emitted by the fluorescent reporter corresponds to a concentration of the fluorescent reporter comprised in the primer. 35. The system according to claim 31, wherein the DNA sequences amplified during a PCR process are identified based on the emitted intensities and spectral wavelengths. 36. The system according to claim 26, wherein the plurality of fluorescent reporters comprised in the plurality of primers are configured to emit fluorescence in a color combination of a plurality of color combinations at an intensity of a plurality of pre-defined intensities. 37. The system according to claim 36, wherein a color combination and pre-defined intensity corresponds uniquely to a particular DNA sequence. 38. The system according to claim 37, wherein the DNA sequences amplified during a PCR process are identified based on the emitted color combinations and intensities. 39. The system according to claim 26, wherein the thermal cycler is configured to cycle the temperature in the plurality of reactor volumes in a plurality of thermocycles. 40. The system according to claim 39, wherein each fluorescent reporter of the plurality of fluorescent reporters is further configured to emit fluorescence in a spectral wavelength after a pre-defined number of thermocycles have elapsed. 41. The system according to claim 40, wherein the pre-defined number of thermocycles after which a fluorescent reporter emits fluorescence corresponds uniquely to a particular DNA sequence of the plurality of DNA sequences. 42. The system according to claim 41, wherein the volume of concentration further comprises a second plurality of primers corresponding to a plurality of DNA sequences and that do not comprise fluorescent reporters. 43. The system according to claim 42, wherein the pre-defined number of thermocycles after which a fluorescent reporter emits fluorescence for a particular DNA sequence corresponds to the ratio of primers from the plurality of primers to the primers from the second plurality of primers. 44. The system according to claim 40, wherein the plurality of primers comprise primers configured bind to DNA sequences at varying efficiencies. 45. The system according to claim 40, wherein a primer of the plurality of primers configured to bind to a DNA sequence with a higher efficiency will emit fluorescence after a lower number of thermocycles has elapsed than a primer configured to bind to a DNA sequence at a lower efficiency. 46. The system according to claim 40, wherein the plurality of primers comprise primers that are configured to bind to a DNA sequence at varying temperatures in the plurality of reactor volumes. 47. The system according to claim 46, wherein the temperature in the plurality of reactor volumes is cycled through a pre-defined range during a thermocycle of the plurality of thermocycles. 48. The system according to claim 40, wherein the DNA sequences amplified during a PCR process are identified based on the numbers of thermocycles elapsed before fluorescences are emitted. 49. The system according to claim 26, wherein the fluorescent reporter comprises an intercalating dye. 50. The system according to claim 26, wherein the fluorescent reporter comprises a probe comprising a fluorophore and a quencher. 51. The system according to claim 50, wherein the problem comprises a TaqMan probe. 52. The system according to claim 26, wherein the plurality of reactor volumes comprises one million reactor volumes.
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