Method and apparatus for generating thermal melting curves in a microfluidic device
First Claim
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1. A method of performing thermal melt analysis of a nucleic acid in a microfluidic device, the method comprising:
- providing a microfluidic device having at least one microfluidic channel, the at least one microfluidic channel having an upstream portion and a downstream portion, introducing fluid comprising the nucleic acid and amplification reagents into the microfluidic channel so that the fluid flows from the upstream portion to the downstream portion of the channel, cycling the temperature in the upstream portion so that the nucleic acid undergoes amplification, subjecting the fluid to a series of temperatures in the downstream portion, wherein the series of temperatures includes a temperature high enough to cause denaturization of the nucleic acid, and measuring a detectable property emanating from the fluid that is indicative of the extent of denaturation of the nucleic acid in the downstream portion.
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Abstract
The present invention provides novel methods and devices that employ microfluidic technology to generate molecular melt curves. In particular, the devices and methods in accordance with the invention are useful in providing for the analysis of PCR amplification products.
282 Citations
65 Claims
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1. A method of performing thermal melt analysis of a nucleic acid in a microfluidic device, the method comprising:
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providing a microfluidic device having at least one microfluidic channel, the at least one microfluidic channel having an upstream portion and a downstream portion, introducing fluid comprising the nucleic acid and amplification reagents into the microfluidic channel so that the fluid flows from the upstream portion to the downstream portion of the channel, cycling the temperature in the upstream portion so that the nucleic acid undergoes amplification, subjecting the fluid to a series of temperatures in the downstream portion, wherein the series of temperatures includes a temperature high enough to cause denaturization of the nucleic acid, and measuring a detectable property emanating from the fluid that is indicative of the extent of denaturation of the nucleic acid in the downstream portion. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45)
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46. A method of determining the value of a physical parameter that corresponds to a reference temperature within a channel in a microfluidic device, the method comprising:
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flowing a fluid comprising a molecule of known Tm through the channel, varying a physical parameter that correlates with the temperature within the channel, generating a thermal property curve for the molecule by measuring the value of a detectable property of the molecule as a function of the parameter; and
determining the values of the detectable property and the parameter at the point in the thermal property curve that corresponds to the Tm of the molecule. - View Dependent Claims (47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61)
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62. A method of determining the thermal inertia of a microfluidic device, the method comprising:
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flowing a fluid comprising a fluorescent material through a channel in a microfluidic device, wherein the amount of fluorescence generated by the fluorescent material varies with temperature, varying the temperature of an external surface of the microfluidic device, wherein the variation comprises a pattern with a recognizable feature, wherein the variation in temperature of the external surface produces a corresponding variation in temperature within the channel, and wherein the variation in temperature in the channel produces a corresponding variation in the amount of fluorescence generated by the fluorescent material, measuring the fluorescence generated by the fluorescent material in the channel, and determining a time offset between the temperature variations of the external surface and the variations in the amount of fluorescent by measuring the time delay between the imposition of the recognizable feature on the external surface and the appearance of the recognizable feature in the fluorescence generated by the fluorescent material, whereby the time offset is indicative of the thermal inertia. - View Dependent Claims (63, 64)
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65. A method of determining whether the fluid in a microchannel is flowing or stagnant, the method comprising:
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applying a range of driving forces to cause fluid to flow through a microchannel at a range of flow rates, wherein the fluid comprises a fluorescent material, and wherein the range of flow rates includes a zero flow rate, exciting the fluorescent material with light of a sufficient intensity to photobleach the fluorescent material, measuring the amount of fluorescence emanating from the fluorescent material in the microchannel at a plurality of flow rates in the range of flow rates, determining the flow rate at which the amount of fluorescence emanating from the fluorescent material is minimized, whereby that flow rate is the zero flow rate.
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Specification