PCR microreactor for amplifying DNA using microquantities of sample fluid
AssigneeAgilent Technologies, Inc.
InventorsJacqueline Tso, Sally A. Swedberg, Paul K. Wolber
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Abstract
A microanalytical device is provided for conducting chemical processes using small amounts of fluid. The devices include microstructures, e.g., microcavities, microchannels and the like, that are laser ablated or otherwise formed in a support substrate, and can be used in a variety of chemical and biochemical methods, including chromatographic, electrophoretic and electrochromatographic separations, screening and diagnostics, and chemical and biochemical synthesis. The devices are formed from a material that is thermally and chemically stable and resistant to biofouling, significantly reducing electroosmotic flow and unwanted adsorption of solute. Preferred materials are polymeric.
Claims51
In a device for conducting the polymerase chain reaction (PCR) process comprising a reaction chamber defined by two or more interior surfaces, a means for introducing PCR reaction components into the chamber, a means for removing the PCR reaction product from the chamber, and a means for controlling the temperature of the reaction chamber, the improvement which comprises:
- fabricating the device from a chemically inert material that does not contain silicon and is thermally, chemically and mechanically stable under the conditions at which a PCR reaction is conducted, and employing a reaction chamber adapted to contain in the range of approximately 1 μl to 500 μl of fluid, wherein the interior surfaces of the reaction chamber are coated with a sorption-reducing polyethylene oxide layer so as to enhance thermal stability, chemical stability and biofouling resistance.
A microreactor for amplifying DNA using the polymerase chain reaction (PCR) process, comprising:
- a substrate having first and second substantially planar opposing surfaces, said substrate having a cavity and at least one microchannel formed in the first planar surface, wherein the cavity serves as a reaction zone that is in fluid communication with each microchannel;
- a cover plate arranged over the first planar surface, said cover plate in combination with the cavity defining a reaction chamber, and with each microchannel defining a microcolumn;
- and
- at least one inlet port and at least one outlet port communicating directly or indirectly with the reaction chamber, said ports enabling the passage of fluid from an external source into and through the reaction chamber, thereby defining a fluid flow path;
- wherein the substrate and the cover plate are comprised of a chemically inert material that does not contain silicon and is thermally stable and resistant to biofouling under the conditions used for conducting PCR amplification of DNA, and the interior surfaces of the reaction chamber are coated with a sorption-reducing polyethylene oxide layer so as to enhance thermal stability, chemical stability and biofouling resistance.
In a method for conducting the polymerase chain reaction (PCR) process to amplify DNA in a sample which comprises heating the sample to separate double-stranded DNA into single-stranded DNA, cooling the sample so as to allow hybridization of primer oligonucleotides to the single-stranded DNA, replicating the DNA using a DNA polymerase, and repeating the aforementioned steps to achieve the desired degree of amplification, the improvement,which comprises:
- conducting the PCR in a microreactor comprised of a chemically inert material that does not contains silicon and is thermally, chemically and mechanically stable under the conditions at which the PCR reaction is conducted, and employing a reaction chamber adapted to contain approximately 1μl to 500 μl of fluid, wherein the interior surfaces of the reaction chamber are coated with a sorption-reducing polyethylene oxide layer so as to enhance thermal stability, chemical stability and biofouling resistance.
A method for amplifying the quantity of a DNA molecule of interest contained in a small volume of sample fluid using the polymerase chain reaction process, comprising:
- (a) introducing into a microreactor up to about 10 μl of a sample fluid containing the DNA molecule of interest in double-stranded form, a first and a second primer molecule complementary to opposing strands of the DNA molecule, a thermostable DNA polymerase, free deoxynucleoside triphosphates and PCR buffer, the microreactor comprising
- a substrate having first and second substantially planar opposing surfaces, said substrate having a cavity formed in the first planar surface, wherein the cavity serves as a reaction zone,
- a cover plate arranged over the first planar surface, said cover plate in combination with the cavity defining a reaction chamber, and
- at least one inlet port and at least one outlet port in fluid communication with the reaction chamber, said ports enabling the passage of sample fluid from an external source into and through the reaction chamber, thereby defining a fluid flow path,
- wherein the substrate and the cover plate are comprised of a chemically inert material that does not contain silicon and is thermally stable and resistant to biofouling, and wherein the interior surfaces of the reaction chamber are coated directly with a sorption-reducing polyethylene oxide layer so as to enhance thermal stability, chemical stability and biofouling resistance;
- (b) applying a motive force to the device to move the sample fluid along the flow path into the reaction chamber;
- (c) heating the sample fluid in the reaction chamber to separate the double-stranded DNA into single-stranded DNA;
- (d) cooling the sample so as to allow hybridization of the primer molecules to opposing strands of the single-stranded DNA and replication of the single-stranded DNA by the DNA polymerase;
- and
- (e) repeating steps (c) and (d) to achieve the desired degree of amplification.
A method for amplifying the quantity of a DNA molecule of interest contained in a small volume of sample fluid using the polymerase chain reaction process, comprising:
- (a) introducing into a microreactor up to about 10 μl of a sample fluid containing the DNA molecule of interest in double-stranded form, a first and a second primer molecule complementary to opposing strands of the DNA molecule, a thermostable DNA polymerase, free deoxynucleoside triphosphates and PCR buffer, the microreactor comprising
- a substrate having first and second substantially planar opposing surfaces, said substrate having a cavity and at least one microchannel formed in the first planar surface, wherein the cavity serves as a reaction zone that is in fluid communication with each microchannel,
- a cover plate arranged over the first planar surface, said cover plate in combination with the cavity defining a reaction chamber, and with each microchannel defining a microcolumn, and
- at least one inlet port and at least one outlet port communicating directly or indirectly with the reaction chamber, said ports enabling the passage of sample fluid from an external source into and through the reaction chamber, thereby defining a fluid flow path,
- wherein the substrate and the cover plate are comprised of a chemically inert material that does not contain silicon and is thermally stable and resistant to biofouling, and the interior surfaces of the reaction chamber are coated with a sorption-reducing polyethylene oxide layer so as to enhance thermal stability, chemical stability and biofouling resistance;
- (b) applying a motive force to the device to move the sample fluid along the flow path into the reaction chamber;
- (c) heating the sample fluid in the reaction chamber to separate the double-stranded DNA into single-stranded DNA;
- (d) cooling the sample so as to allow hybridization of the primer molecules to opposing strands of the single-stranded DNA and replication of the single-stranded DNA by the DNA polymerase;
- and
- (e) repeating steps (c) and (d) to achieve the desired degree of amplification.
Dependencies are inferred from the claim text.