# US 2010/0167288 A1 · Methods and Compositions Related to Continuous Flow Thermal Gradient PCR > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 2010/0167288 A1 - Document ID: US20100167288A1 - Status: Published application - Kind: A1 - Application number: 12/514,671 - Filed: 2007-11-14 - Published: 2010-07-01 - Earliest claimed: 2006-11-14 - Assignees: University of Utah Research Foundation - Inventors: Bruce Kent Gale; Niel Davenport Crews; Carl Thomas Wittwer - Web page: https://patentgrep.com/patent/US20100167288A1 ## CPC - B01L 3/502707: {characterised by the manufacture of the container or its components} - B01L 7/525: {with physical movement of samples between temperature zones} - B01L 7/54: {using spatial temperature gradients} - G01N 21/6428: {Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" (in vivo A61B5/00; immunoassay G01N33/53)} - G01N 21/6456: {Spatial resolved fluorescence measurements; Imaging} - B01L 2300/0816: Cards, e.g. flat sample carriers usually with flow in two horizontal directions - B01L 2300/0861: Configuration of multiple channels and/or chambers in a single devices - B01L 2300/1822: using Peltier elements - B01L 2300/1827: using resistive heater - B01L 2400/0487: fluid pressure, pneumatics - Y10T 156/1056: Perforating lamina - Y10T 156/1062: Prior to assembly - B01L: CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE - G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q) - Y10T: TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION ## Abstract Disclosed are compositions and a method for amplification and detection of nucleic acid sequences based on continuous flow thermal gradient PCR. ## Claims ### Claim 1 A device for replicating nucleic acid, said device comprising: a microchannel extending from an inlet port to an outlet port; and a heater or heaters for producing a spatial temperature gradient. ### Claim 2 (depends on 1) The device of claim 1, wherein the microchannel forms a serpentine pattern over the temperature gradient. ### Claim 3 (depends on 1) The device of claim 1, wherein the temperature gradient allows for gradual heating of the sample. ### Claim 4 (depends on 1) The device of claim 1, wherein the temperature gradient is a steady-state gradient. ### Claim 5 (depends on 1) The device of claim 1, wherein the heater or heaters is disposed at the far edges of the device, away from the microchannel. ### Claim 6 (depends on 1) The device of claim 1, wherein the heater is disposed along a centerline of the microchannel. ### Claim 7 (depends on 1) The device of claim 1, wherein the device also comprises a cooling component. ### Claim 8 (depends on 7) The device of claim 7, wherein the cooling component cools by air. ### Claim 9 (depends on 7) The device of claim 7, wherein the cooling component cools by using cooling fins. ### Claim 10 (depends on 7) The device of claim 7, wherein the cooling component is a thermoelectric cooler. ### Claim 11 (depends on 1) The device of claim 1, wherein the spatial temperature gradient is between approximately 1° C. and 50° C. or more per millimeter. ### Claim 12 (depends on 7) The device of claim 7, further comprising a pump for pumping fluid through the microchannel. ### Claim 13 (depends on 1) The device of claim 1, wherein the microchannel has a heating portion and a cooling portion. ### Claim 14 (depends on 13) The device of claim 13, wherein each portion of the microchannel has a width, wherein the width of the heating portion is wider than the width of the cooling portion. ### Claim 15 (depends on 13) The device of claim 13, wherein each portion of the microchannel has a width, wherein the width of the heating portion is narrower than the width of the cooling portion. ### Claim 16 (depends on 1) The device of claim 1, further having a light source for emitting light to thereby cause fluorescence. ### Claim 17 (depends on 16) The device of claim 16, further comprising a sensor for measuring the spatial distribution of fluorescence. ### Claim 18 (depends on 1) The device of claim 1, wherein the microchannel is formed in a thin film sandwiched between two plates. ### Claim 19 (depends on 1) The device of claim 1, wherein the microchannel has a channel depth of between 10 and 200 μm. ### Claim 20 (depends on 11) The device of claim 11, wherein the cooling portion of the microchannel has a width of approximately 30-500 μm. ### Claim 21 (depends on 1) The device of claim 1, wherein the heating portion of the microchannel has a width of approximately 5-5000 μm. ### Claim 22 (depends on 1) The device of claim 1, wherein the microchannel performs between 20 and 50 amplification cycles. ### Claim 23 (depends on 1) The device of claim 1, wherein the microchannel has a length of approximately 1 to 100 centimeters. ### Claim 24 (depends on 1) The device of claim 1, wherein the microchannel is formed using a Xurography process. ### Claim 25 (depends on 1) The device of claim 1, wherein the microchannel is formed using a wet etching process. ### Claim 26 (depends on 1) The device of claim 1, wherein the heater or heaters is maintained at a steady temperature. ### Claim 27 A device for replicating a nucleic acid, said device comprising: a microchannel; two plates; and a heater; wherein the microchannel is sandwiched between the two plates and the heater is operable to form a spatial temperature gradient across the microchannel. ### Claim 28 (depends on 27) The device of claim 27, wherein the microchannel is comprised of a thin film. ### Claim 29 (depends on 28) The device of claim 28, wherein the microchannel is created using at least one of Xurography and glass etching. ### Claim 30 (depends on 28) The device of claim 28, wherein the microchannel has a serpentine pattern. ### Claim 31 (depends on 28) The device of claim 28, further comprising at least one fastener for holding the two plates together. ### Claim 32 A device for replicating nucleic acid, said device comprising: a channel having a plurality of sections forming a continuous pattern; and a heater disposed along a centerline of the continuous pattern; wherein each section of the channel comprises a first portion and a second portion, the first portion of the channel being narrower than the second portion of the channel. ### Claim 33 (depends on 32) The device of claim 32, wherein the wide and narrow portions are determined by the heating and cooling sections of the device. ### Claim 34 (depends on 33) The device of claim 33, wherein the dimensions of the device determine the velocity of the fluid therein, thereby controlling the temperature ramp rates. ### Claim 35 (depends on 34) The device of claim 34, wherein the continuous pattern is a serpentine pattern. ### Claim 36 (depends on 34) The device of claim 34, further comprising a pump for pumping fluid through the channel. ### Claim 37 A method of amplifying a nucleic acid, the method comprising the steps of: a) forming a steady state temperature gradient on a device comprising microchannels; and b) exposing a nucleic acid to the temperature gradient in a manner conducive for amplification; thereby amplifying a nucleic acid. ### Claim 38 (depends on 37) The method of claim 37, wherein the microchannel is in a serpentine pattern. ### Claim 39 (depends on 37) The method of claim 37, wherein the nucleic acid is amplified multiple times. ### Claim 40 A method for monitoring nucleic acid replication using a microchip, said method comprising the steps of: a) forming a temperature gradient across a device; and b) exposing a nucleic acid to the temperature gradient in a manner conducive for amplification; and c) detecting nucleic acid amplification using fluorescent monitoring; thereby monitoring nucleic acid amplification using a microchip. ### Claim 41 (depends on 40) The method of claim 40, wherein the nucleic acid is detected by exposing the nucleic acid to a dye, then detecting interaction of the dye and the nucleic acid. ### Claim 42 (depends on 41) The method of claim 41, wherein the dye is fluorescent. ### Claim 43 (depends on 41) The method of claim 41, wherein the dye is intercalating. ### Claim 44 (depends on 40) The method of claim 40, wherein each cycle of nucleic acid replication can be detected. ### Claim 45 (depends on 40) The method of claim 40, wherein amplification can be detected with a single photograph. ### Claim 46 (depends on 44) The method of claim 44 further comprising the step of measuring the amount of fluorescence produced by the dye after each extension. ### Claim 47 (depends on 46) The method of claim 46, wherein amplification is detected by monitoring the channel-wise growth in fluorescence and wherein the melting behavior of the amplicon is detected during each denaturing process. ### Claim 48 (depends on 47) The method of claim 47, further comprising the step of providing the information in real time. ### Claim 49 (depends on 40) The method of claim 40 wherein the nucleic acid is pumped into the microchannel. ### Claim 50 (depends on 49) The method of claim 49 wherein the nucleic acid is pumped into the microchannel using continuous flow. ### Claim 51 (depends on 40) The method of claim 40, wherein detecting nucleic acid further comprises determining information related to the denaturing or melting of the double stranded nucleic acid. ### Claim 52 (depends on 41) The method of claim 41, wherein the dye is at least one of SYBR Green, LC Green, and LC Green Plus. ### Claim 53 (depends on 47) The method of claim 47, wherein a melting curve analysis is conducted on the nucleic acid. ### Claim 54 (depends on 40) The method of claim 40, wherein more than one nucleic acid sample is amplified at a time. ### Claim 55 (depends on 54) The method of claim 54, wherein the nucleic acid samples differ in sequence. ### Claim 56 (depends on 55) The method of claim 55, wherein an analysis of the spatial fluorescence and temperature distribution can distinguish between the multiple samples of nucleic acids. ### Claim 57 A method of forming a chip with microchannels for use in continuous-flow PCR, said method comprising the steps of: a) creating a digital image of the microchannels using a digital computing machine; b) sending the digital image of the microchannels to a plotting device such that the plotting device forms the microchannels on a thin film; and c) sandwiching the thin film between two plates; thereby forming a chip with microchannels for use in continuous-flow PCR. ### Claim 58 (depends on 57) The method of claim 57, wherein the digital image of the microchannels includes a serpentine pattern. ### Claim 59 (depends on 57) The method of claim 57, further comprising the step of pre-drilling holes in the plates. ### Claim 60 (depends on 57) The method of claim 57, further comprising the step of applying pressure to the two plates. ### Claim 61 (depends on 57) The method of claim 57, further comprising the step of pre-coating the thin film with an adhesive. ### Claim 62 (depends on 57) The method of claim 57, further comprising the step of curing the two plates and the thin film at an elevated temperature. ### Claim 63 (depends on 56) The method of claim 56, further comprising the step of forming fluid interconnects in at least one of the two plates. ### Claim 64 (depends on 56) The method of claim 56, further comprising the step of forming a spatial temperature gradient. ### Claim 65 (depends on 56) The method of claim 56, further comprising the step of pumping a liquid having DNA therein through the microchannels. ### Claim 66 (depends on 65) The method of claim 65, further comprising pumping the liquid at a constant volume flow rate. ### Claim 67 (depends on 65) The method of claim 65, further comprising varying the width of the microchannels to control temperature ramping rates.