METHOD AND APPARATUS FOR APPLYING CONTINUOUS FLOW AND UNIFORM TEMPERATURE TO GENERATE THERMAL MELTING CURVES IN A MICROFLUIDIC DEVICE

Abstract

The present invention provides a method for performing thermal melt analysis using a microfluidic device, the method comprising providing a microfluidic device having at least one microfluidic channel, introducing fluid comprising into the at least one microfluidic channel, continuously flowing the fluid through the at least one microfluidic channel while varying the temperature of the entire fluid stream as it moves through the at least one microfluidic channel by uniformly heating the entire fluid stream, and measuring, while continuously flowing the fluid through the at least one microfluidic channel, a detectable property emanating from the fluid,

Description

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a schematic of a system comprising a computer, detector and temperature controller.

FIGS. 2A-2B are exemplary illustrations of two microfluidic systems capable of performing continuous flow format melting curve analysis according to the present invention.

FIG. 3 depicts an example of a microfluidic device known in the art in which the present invention can be implemented.

FIGS. 4A-4D depict portions of an integrated system that interfaces with the microfluidic device of FIG. 3

FIGS. 5A and 5B depict example data obtained using the present invention in conjunction with the microfluidic device of FIG. 3 and the integrated system of FIGS. 4A-4D.

Claims

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,introducing fluid comprising the nucleic acid and reagents into the at least one microfluidic channel;continuously flowing the fluid through the at least one microfluidic channel while varying the temperature of the fluid stream as it moves through the at least one microfluidic channel by uniformly heating the fluid stream; andmeasuring, while continuously flowing the fluid through the at least one microfluidic channel, a detectable property emanating from the fluid, wherein the detectable property indicates an extent of denaturation of the nucleic acid.

2. The method of claim 1, wherein the nucleic acid is DNA.

3. The method of claim 1, wherein the reagents comprise primers, a thermostable polymerase, nucleotides, and fluorescent dyes.

4. The method of claim 1, wherein the temperature of the fluid stream is continuously increased at a rate in the range of 0.1° C./second to 1° C./second.

5. The method of claim 1, wherein the temperature of the fluid stream is continuously increased at a rate in the range of 0.01° C./second to 0.1° C./second.

6. The method of claim 1, wherein the temperature of the fluid stream is continuously increased at a rate in the range of 1° C./second to 10° C./second.

7. The method of claim 1, wherein the temperature of the fluid stream is uniformly heated using joule heating.

8. The method of claim 1, wherein the temperature of the fluid stream is uniformly heated using non-joule heating.

9. The method of claim 1, wherein the non-joule heating comprises heating comprises heating the at least one microfluidic channel using a thermal block in thermal contact with the at least one microfluidic channel.

10. The method of claim 1, wherein the thermal block maintains a uniform temperature along the length of the at least one microfluidic channel.

11. The method of claim 1, wherein the uniform temperature is generated by placing a plurality of locations of the thermal block in thermal contact with a plurality of peltier devices respectively, wherein the plurality of peltier devices are controlled to the same temperature.

12. The method of claim 1, wherein a uniform temperature is maintained along the length of the at least one microfluidic channel by maintaining a constant cross-section of the length of the at least microfluidic channel and passing an electric current through the fluid flowing through the at least one microfluidic channel

13. The method of claim 1, wherein the detectable property comprises fluorescence.

14. The method of claim 13, wherein the fluorescence is generated by FRET or a molecular beacon.

15. The method of claim 13, wherein the fluorescence is generated by a fluorescent dye, and wherein the amount of fluorescence generated by the fluorescent dye is indicative of the extent of thermal denaturation of the nucleic acid.

16. The method of claim 15, wherein the fluorescent dye is an intercalating dye.

17. The method of claim 15, wherein the fluorescent dye is ethidium bromide.

18. The method of claim 15, wherein the fluorescent dye is a minor groove binding dye.

19. The method of claim 15, wherein the fluorescent dye is a SYBR green dye.

20. The method of claim 1, wherein measuring the detectable property comprises measuring the detectable property at at least one location along the length of the at least one microfluidic channel, whereby the detectable property emanating from the fluid at a plurality of temperatures is measured.

21. The method of claim 1, wherein the detectable property is fluorescence polarization.

22. The method of claim 1, wherein the detectable property is UV absorbance.

23. The method of claim 1, wherein the detectable property is selected from the group consisting of heat capacity, electrical resistance, and dielectric properties.