Real-time PCR in micro-channels
First Claim
Patent Images
1. A method of performing real-time PCR comprising the steps of:
- a) continuously moving a bolus of test solution containing real-time PCR reagents in a channel;
b) moving a carrier fluid in said channel, sequentially alternating with a test bolus;
c) cycling the temperature in a defined section of the channel in order to achieve PCR; and
d) measuring the intensity of the fluorescent signal at a plurality of locations along said defined section of said channel.
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Abstract
The present invention relates to methods for amplifying nucleic acids in micro-channels. More specifically, the present invention relates to methods for performing a real-time polymerase chain reaction (PCR) in a continuous-flow microfluidic system and to methods for monitoring real-time PCR in such systems.
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Citations
48 Claims
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1. A method of performing real-time PCR comprising the steps of:
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a) continuously moving a bolus of test solution containing real-time PCR reagents in a channel; b) moving a carrier fluid in said channel, sequentially alternating with a test bolus; c) cycling the temperature in a defined section of the channel in order to achieve PCR; and d) measuring the intensity of the fluorescent signal at a plurality of locations along said defined section of said channel. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18)
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19. A method for monitoring the progress of a polymerase chain reaction in a channel, comprising the steps of:
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a) moving a bolus of test solution containing real-time PCR reagents in a channel; b) moving a carrier fluid in said channel, sequentially alternating with a test bolus; c) cycling the temperature in a defined section of the channel in order to achieve PCR; d) capturing an image of a reaction-dependent fluorescence signal along a section of the channel; e) measuring the average flow speed in the channel; and f) relating position of the test bolus to the number of temperature cycles experienced by a test bolus from the average flow speed. - View Dependent Claims (20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32)
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33. A method for monitoring the progress of a polymerase chain reaction in a channel, comprising the steps of:
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a) moving a bolus of test solution containing real-time PCR reagents in a channel; b) moving a carrier fluid in the same channel, sequentially alternating with a test bolus; c) applying spatial temperature zones along the channel to achieve PCR; d) monitoring the fluorescence signal at fixed spatial locations along the channel corresponding to fixed points in the PCR cycle; e) measuring the average flow speed of the fluid in the channel; and f) adjusting the flow rate in order to control the timing of the PCR cycle.
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34. A system for performing real-time PCR in a microfluidic device, said system comprising:
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a microfluidic device comprising at least one channel configured to contain at least one test solution containing real-time PCR reagents and at least one carrier fluid; a thermal transfer element integral with or proximal to the microfluidic device, said thermal transfer element configured to thermocycle the test solution along a defined section of the at least one channel for performing real-time PCR in the test solution; a source of illumination integral with or proximal to the microfluidic device and configured to illuminate the at least one channel; and a detector integral with or proximal to the microfluidic device, said detector configured to detect a fluorescent signal at a plurality of locations along said defined section of said at least one channel. - View Dependent Claims (35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48)
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Specification