Methods for Rapid Multiplexed Amplification of Target Nucleic Acids

Abstract

A fast, multiplexed PCR system is described that can rapidly generate amplified nucleic acid products, for example, a full STR profile, from a target nucleic acid. Such systems include, for example, microfluidic biochips and a custom built thermal cycler, which are also described. The resulting STR profiles can satisfy forensic guidelines for signal strength, inter-loci peak height balance, heterozygous peak height ratio, incomplete non-template nucleotide addition, and stutter.

We claim: 
     
         1 . A thermal cycler comprising:
 a temperature control element (TCE), wherein a first surface of said TCE is adapted to receive a sample chamber containing a solution and a sensing chamber containing a thermosensor,   wherein said thermosensor provides feedback to said TCE to set or maintain the solution at a desired temperature.   
     
     
         2 . A thermal cycler comprising:
 a temperature control element (TCE), wherein a first surface of said TCE is adapted to receive a sample chamber containing a solution and a sensing chamber containing a first thermosensor,   wherein said thermosensor provides feedback to said TCE to set or maintain the solution at a desired temperature, further comprising a second thermosensor positioned to monitor the temperature of said first surface of said TCE.   
     
     
         3 . A thermal cycler comprising:
 a temperature control element (TCE), wherein a first surface of said TCE is adapted to receive a sample chamber, said chamber containing a solution and a thermosensor, wherein said thermosensor provides feedback to said TCE to set or maintain the solution at a desired temperature.   
     
     
         4 . The thermal cycler of  claim 2 , further comprising a chip compression element (CCE) positioned over the first surface of the TCE allowing insertion of a substrate between the CCE and the TCE, wherein the CCE provides for thermal communication between the TCE and the substrate. 
     
     
         5 . The thermal cycler of  claim 4 , wherein the CCE comprises a low thermal mass and insulating material. 
     
     
         6 . The thermal cycler of  claim 4 , wherein the CCE is a member of the group consisting of a foam pad, one or a plurality of clips and an air bladder. 
     
     
         7 . The thermal cycler of  claim 2  wherein the first surface of the TCE is adapted to receive a thin-walled tube. 
     
     
         8 . The thermal cycler of  claim 2  having a heating or cooling rate at the first surface of the TCE of about 4-150° C. per second. 
     
     
         9 . The thermal cycler of  claim 2 , further comprising a heat sink comprising a variable speed cooling fan for controlling the temperature of the heat sink. 
     
     
         10 . The thermal cycler of  claim 9 , wherein the heat sink further comprises a second thermocycler for controlling the temperature of the heat sink. 
     
     
         11 . The thermal cycler of  claim 2 , having a temperature stability of about +/−1.0° C. at a sample in a substrate in thermal communication with the first surface of the TCE. 
     
     
         12 . The thermal cycler of  claim 2 , wherein the TCE comprises a high heating and cooling capacity heat pump or a high power output Peltier device. 
     
     
         13 . The thermal cycler of  claim 9 , wherein the heat sink is a fan-cooled heat sink with copper bases and cooling fins. 
     
     
         14 . The thermal cycler of  claim 13 , wherein the heat sink has a thermal resistance of approximately 0.4° C./W or less. 
     
     
         15 . A system comprising
 a biochip comprising one or a plurality of reaction chambers, wherein
 each reaction chamber comprises a microfluidic inlet channel and a microfluidic outlet channel, wherein each reaction chamber is less than 200 μm from a contact surface of the biochip substrate; 
   and a thermal cycler, comprising   a temperature control element (TCE), wherein a first surface of said TCE is adapted to receive a sample chamber containing a solution and a sensing chamber containing a thermosensor, wherein said thermosensor provides feedback to said TCE to set or maintain the solution at a desired temperature,   in thermal communication with the contact surface of the biochip substrate.   
     
     
         16 . A system comprising
 a biochip comprising one or a plurality of reaction chambers, wherein
 each reaction chamber comprises a microfluidic inlet channel and a microfluidic outlet channel, wherein each reaction chamber is less than 100 μm from a contact surface of the biochip substrate; 
   and a thermal cycler, comprising   a temperature control element (TCE), wherein a first surface of said TCE is adapted to receive a sample chamber containing a solution and a sensing chamber containing a thermosensor, wherein said thermosensor provides feedback to said TCE to set or maintain the solution at a desired temperature, further comprising a second thermosensor positioned to monitor the temperature of said first surface of said TCE,   in thermal communication with the contact surface of the biochip substrate.   
     
     
         17 . The system of  claim 16  wherein the microfluidic inlet channel, the microfluidic outlet channel, or both has a via. 
     
     
         18 . The system of  claim 16 , wherein the thermal cycler further comprises a chip compression element (CCE) positioned over the first surface of the TCE allowing insertion of a substrate between the CCE and the TCE, wherein the CCE provides for thermal communication between the TCE and the substrate. 
     
     
         19 . The system of  claim 16 , wherein the chip compression element comprises a low thermal mass and insulating material. 
     
     
         20 . The system of  claim 18 , wherein the CCE is selected from the group consisting of a foam pad, one or a plurality of clips and an air bladder. 
     
     
         21 . The system of  claim 18 , wherein the CCE provides about 5 to about 50 psi of pressure to hold the contact surface of the biochip substrate in thermal contact with the first surface of the TCE. 
     
     
         22 . The system of  claim 16 , wherein the reaction chambers are not coated with a polymer or silane coating or BSA. 
     
     
         23 . The system of  claim 16 , wherein thermal communication between the contact surface of the biochip substrate and the first surface of the TCE is provided in the absence of a thermal coupling solution. 
     
     
         24 . The system of  claim 16 , having a heating or cooling rate at the first surface of the TCE of about 4-150° C. per second. 
     
     
         25 . The system of  claim 16 , wherein the thermal cycler is capable of a heating or cooling rate within the reaction chambers of about 4-150° C. per second. 
     
     
         26 . The system of  claim 16 , further comprising a fan-cooled heat sink with copper bases and cooling fins. 
     
     
         27 . The system of  claim 16 , further comprising a heat sink with a thermal resistance of approximately 0.4° C./W or less. 
     
     
         28 . The system of  claim 16 , further comprising a heat sink with a variable speed cooling fan for controlling the temperature of the heat sink. 
     
     
         29 . The system of  claim 16 , further comprising a heat sink further comprising a second heating element for controlling the temperature of the heat sink. 
     
     
         30 . The system of  claim 16 , wherein the thermal cycler is capable of a temperature stability of +/−1.0° C. at a sample in the biochip. 
     
     
         31 . The system of  claim 16 , wherein the TCE comprises a high heating and cooling capacity heat pump or a high power output Peltier device. 
     
     
         32 . The system of  claim 16 , wherein the biochip substrate is constructed of an organic material, an inorganic material, a crystalline material or an amorphous material. 
     
     
         33 . The system of  claim 16 , wherein the biochip substrate comprises a plastic material. 
     
     
         34 . The system of  claim 33 , wherein the biochip substrate comprises a cyclic olefin co-polymer (COC). 
     
     
         35 . The system of  claim 16 , wherein the biochip substrate comprises 8-128 microfluidic systems. 
     
     
         36 . The system of  claim 16 , wherein each reaction chamber has a volume of less than about 100 μL. 
     
     
         37 . A method for simultaneously amplifying of a plurality of loci in a nucleic acid solution comprising
 providing one or a plurality of reaction chambers wherein
 each reaction chamber comprises 
 (i) a nucleic acid solution comprising at least one copy of at least one target nucleic acid to be amplified; 
 (ii) one or more buffers; 
 (iii) one or more salts; 
 (iv) a primer set corresponding to each of the plurality of loci to be amplified; 
 (v) a nucleic acid polymerase; and 
 (vi) nucleotides, 
   sequentially thermally cycling the temperature of the nucleic acid solution in each reaction chamber between a denaturing state, an annealing state, and an extension state for a predetermined number of cycles at heating and a cooling rates of about 4-150° C./sec, to yield a plurality of amplified loci in each reaction chamber in about 97 minutes or less.   
     
     
         38 . The method of  claim 37 , further comprising
 holding the one or a plurality of reaction solutions at a final state to provide one or a plurality of amplified nucleic acid products.   
     
     
         39 . A method for simultaneously amplifying of a plurality of loci in a nucleic acid solution comprising
 providing one or a plurality of reaction chambers wherein
 each reaction chamber comprises 
 (i) a nucleic acid solution comprising at least one copy of at least one target nucleic acid to be amplified; 
 (ii) one or more buffers; 
 (iii) one or more salts; 
 (iv) a primer set corresponding to each of the plurality of loci to be amplified; 
 (v) a nucleic acid polymerase; and 
 (vi) nucleotides, 
   sequentially thermally cycling the temperature of the nucleic acid solution in each reaction chamber for a predetermined number of cycles at heating and a cooling rates of about 4-150° C./sec, to yield a plurality of amplified loci in each reaction chamber in about 97 minutes or less.   
     
     
         40 . A method for simultaneously amplifying 5 or more loci in a nucleic acid solution comprising
 providing one or a plurality of reaction chambers wherein
 each reaction chamber comprises 
 (i) a nucleic acid solution comprising at least one copy of at least one target nucleic acid to be amplified; 
 (ii) one or more buffers; 
 (iii) one or more salts; 
 (iv) a primer set corresponding to the 5 or more loci to be amplified; 
 (v) a nucleic acid polymerase; and 
 (vi) nucleotides, 
   sequentially thermally cycling the temperature of the nucleic acid solution in each reaction chamber between a denaturing state, an annealing state, and an extension state for a predetermined number of cycles at heating and a cooling rates of about 4-150° C./sec, to yield 5 or more amplified loci in each reaction chamber.   
     
     
         41 . The method of  claim 40  wherein said nucleic acid solution is present in a biochip. 
     
     
         42 . The method of  claim 40  wherein said nucleic acid solution is present in a thin walled tube. 
     
     
         43 . The method of  claim 40 , wherein the 5 or more amplified loci are produced in less than about 97 minutes. 
     
     
         44 . The method of  claim 40 , wherein the amplified loci are produced in less than about 45 minutes. 
     
     
         45 . The method of  claim 40  further comprising, prior to the sequential thermal cycling,
 heating the one or a plurality of reaction solutions to a first temperature suitable for hot-start activation of the nucleic acid polymerases; and 
 holding the one or a plurality of reaction solutions at the first temperature for a first period of time suitable for hot-start activation of the nucleic acid polymerases. 
 
     
     
         46 . The method of  claim 45 , wherein the first period of time is less than about 90 seconds. 
     
     
         47 . The method of  claim 45  wherein the first temperature is about 90 to about 99° C. 
     
     
         48 . The method of  claim 45 , wherein the thermal cycling is provided by a thermal cycler of  claim 1 . 
     
     
         49 . The method  claim 44 , wherein the nucleic acid polymerase has an extension rate of at least 100 bp/sec. 
     
     
         50 . The method of any one of  claim 44 , wherein each reaction chamber has a volume of less than about 100 μL. 
     
     
         51 . The method of any one of  claim 44 , wherein each reaction chamber is separated from a thermal cycler by less than about 200 μm. 
     
     
         52 . The method of any one of  claim 44 , wherein each nucleic acid solution comprises about 1 to about 1000 copies of a target nucleic acid. 
     
     
         53 . The method  claim 44 , wherein the nucleic acid polymerase is SpeedSTAR, PHUSION, Hot MasterTaq™, PHUSION Mpx, PyroStart, KOD, Z-Taq, or CS3AC/LA. 
     
     
         54 . The method of  claim 44 , wherein analysis of each of the amplified nucleic acid products satisfies forensic interpretation guidelines. 
     
     
         55 . The method of  claim 44 , wherein the denaturing state is about 95° C. for about 4 seconds. 
     
     
         56 . The method of  claim 44 , wherein the annealing state is about 59° C. for about 15 seconds. 
     
     
         57 . The method of  claim 44 , wherein the extension state is about 72° C. for about 7 seconds. 
     
     
         58 . The method  claim 44 , wherein the final state is about 70° C. for about 90 seconds. 
     
     
         59 . The method  claim 44 , wherein the one or a plurality of reaction solutions are cooled from the denaturing state to the annealing state at a first cooling rate of about 10 to about 50° C./sec. 
     
     
         60 . The method of  claim 44 , wherein the one or a plurality of reaction solutions are heated from the annealing state to the extension state at a first heating rate of about 10 to about 50° C./sec. 
     
     
         61 . The method  claim 44 , wherein the one or a plurality of reaction solutions are heated from the extension state to the denaturing state at a second heating rate of about 10 to about 50° C./sec. 
     
     
         62 . The method of  claim 44 , wherein the one or a plurality of amplified nucleic acid products are obtained in about 10 to about 90 minutes. 
     
     
         63 . The method of  claim 44 , wherein each reaction solution comprises about 0.005 to about 10 ng of a target nucleic acid. 
     
     
         64 . The method of  claim 44 , wherein the target nucleic acid comprises a human nucleic acid, microbial nucleic acid, or viral nucleic acid. 
     
     
         65 . The method of  claim 44 , wherein 10 to 250 loci are simultaneously amplified. 
     
     
         66 . The method of  claim 65 , wherein the loci comprise amelogenin, D8S1179, D21S11, D7S820, CFS1PO, D3S1358, TH01, D13S317, D16S539, D2S1338, D19S433, vWA, TPOX, D18S51, D5S818, FGA, or a plurality thereof. 
     
     
         67 . The method of  claim 44 , wherein the predetermined number of cycles is between about 10 and about 50 cycles. 
     
     
         68 . The method of  claim 44 , wherein one or a plurality of thin-wall reaction tubes comprise the one or a plurality of reaction chambers. 
     
     
         69 . An integrated biochip system comprising
 a biochip comprising at least two reaction chambers in microfludic communication, wherein a first reaction chamber is in thermal communication with a thermal cycler, comprising:   a temperature control element (TCE), wherein a first surface of said TCE is adapted to receive a sample chamber containing a solution and a sensing chamber containing a thermosensor, wherein said thermosensor provides feedback to said TCE to set or maintain the solution at a desired temperature,   wherein a contact surface of the biochip is in thermal communication with the first surface of the thermal cycler;   and   a second reaction chamber in fluid connection with the first reaction chamber and adapted for   (i) nucleic acid extraction;   (ii) nucleic acid purification;   (iii) pre-PCR nucleic acid cleanup;   (iv) post-PCR cleanup;   (v) pre-sequencing cleanup;   (vi) sequencing;   (vii) post-sequencing cleanup;   (viii) nucleic acid separation;   (ix) nucleic acid detection;   (x) reverse transcription;   (xi) pre-reverse transcription cleanup;   (xii) post-reverse transcription cleanup;   (xiii) nucleic acid ligation;   (xiv) nucleic acid hybridization;   (xv) quantification   wherein the first reaction chamber is less than 200 μm from a contact surface of the biochip.