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.

1 - 69  (canceled) 
     
     
         70 . A method for simultaneously amplifying of a plurality of loci in a nucleic acid solution comprising:
 providing in a single solutions contained in at least two reaction chambers located in a biochip, samples having at least ten target nucleic acid loci to be amplified, with at least ten different primer pairs, each primer pair hybridizing to one of the at least ten loci to be amplified, said solution further comprising:   (i) one or more buffers;   (ii) one or more salts;   (iii) a nucleic acid polymerase; and   (iv) nucleotides; and   providing a thermal control system comprised of (i) a TCE having a first surface in thermal communication with said at least two reaction chambers, said TCE further comprising a means for heating and cooling, (ii) at least one thermosensor, (iii) a controller that receives signals from said at least one thermosensor and (iv)a power supply, said at least one thermosensor positioned and configured to measure the effective temperature of each of the single solutions in the reaction chambers of the biochip and to provide feedback to the TCE to heat or cool the solution to set or maintain the solution at a desired temperature;   obtaining, with said controller of the thermal control system, a target sample solution temperature, from a control algorithm that stores the target sample temperature, for a sample for a first sub-step of a plurality of predetermined processing sub-steps, to be performed on the samples, where in the first sub-step of the processing step is one of a denaturing, an annealing or an extending of a replication of a polymerase chain reaction;   obtaining with said controller from said control algorithm, a time and temperature profile representing a comparison between the target sample temperature as measured by said thermosensor in thermal communication with said at least two reaction chambers, and a plurality of different temperatures for said TCE, wherein the plurality of different temperatures are different from each other and different from the target sample temperature and correspond to different time points, of a predetermined set of time points, of the first sub-step;   identifying, with the controller from said control algorithm, for a first time point of the different time points for the first sub-step, a first temperature of the plurality of different temperatures using the time and temperature profile; and   controlling, with the controller from said control algorithm, a temperature of the TCE at the first time point of the first sub-step to be the first temperature, wherein the TCE is in thermal communication with said at least two reaction chambers and controls a temperature of said samples.   
     
     
         71 . The method of  claim 70 , further comprising:
 identifying, for a second time point of the different time points for the first sub-step, a second temperature of the plurality of different temperatures using the time and temperature profile; and   controlling the temperature of the TCE at the second time point of the sub-step to be the second temperature.   
     
     
         72 . The method of  claim 71 , further comprising:
 maintaining a temperature of at least one reaction chamber at approximately a same temperature for the first and second time points of the sub-step by changing the temperature of the TCE from the first temperature at the first time point to the second temperature at the second time point.   
     
     
         73 . The method of  claim 71  further comprising:
 identifying, for at least one additional time point of the different time points, at least one additional temperature of the plurality of different temperatures using the time and temperature profile and the plurality of different temperatures and the at least one additional time point; and 
 controlling the temperature of the TCE at the at least one additional time point of the sub-step to be the at least one additional temperature. 
 
     
     
         74 . The method of  claim 73  wherein the controlling of the temperature includes gradually changing the temperature from the second temperature to the at least one additional temperature. 
     
     
         75 . The method of  claim 73  further comprising:
 maintaining a temperature of the at least one reaction chamber at approximately a same temperature for the first, the second and the at least one additional time points of the sub-step by changing the temperature of the TCE from the first temperature at the first time point to the second temperature at the second time point to the at least one additional temperature at the at least one additional time point. 
 
     
     
         76 . The method of  claim 75  wherein maintaining the temperature of the at least one reaction chamber maintains an approximately constant temperature of the sample. 
     
     
         77 . The method of  claim 76  wherein the approximately constant temperature is a temperature from a group consisting of 95 degrees; 59 degrees; and 72 degrees Celsius. 
     
     
         78 . The method of  claim 70  wherein the processing step is a DNA sequencing step. 
     
     
         79 . The method of  claim 70 , wherein for each of the denaturing, the annealing and the extending of replication, the temperature of the sample is approximately constant. 
     
     
         80 . The method of  claim 70 , further comprising:
 controlling, with the controller, the temperature of the TCE to set a temperature of a reaction chamber at the target sample temperature;   measuring with the at least one thermosensor positioned and configured to measure the effective temperatures of each of the single solutions in the reaction chambers, a temperature of the at least two reaction chambers concurrently with controlling the temperature of the TCE which results in the temperature of each of the single solutions in the reaction chambers being at the target sample temperature.   
     
     
         81 . The method of  claim 70  further comprising:
 obtaining at least one additional target sample temperature wherein the time and temperature profile provides at least one additional target sample temperature and at least one additional set of temperatures of the TCE for at least one additional sub-step of the processing step; 
 identifying, for a time point of the at least one additional sub-step, a temperature of the at least one additional set of temperatures using at least one additional time and temperature profile generated using the at least one additional target sample temperatures and the at least one set of additional temperatures and the time point of the at least one additional sub-set; and 
 controlling the temperature of the TCE at the time point of the at least one additional sub-set at the at least one additional set of temperatures. 
 
     
     
         82 . An apparatus comprising:
 a controller of a thermal control system configured to:   obtain a target sample temperature, from a controller that stores the target sample temperature, for a sample for a first sub-step, of a plurality of predetermined sub-steps, of a processing step, of a plurality of predetermined processing steps, to be performed on the sample, wherein the first sub-step of the processing step is one of a denaturing, an annealing or an extending of a replication of a polymerase chain reaction sub-step;   obtain a time and temperature profile, from the controller wherein the profile provides a correlation between the target sample temperature, as measured by a thermosensor in thermal communication with a reaction chamber, with a target sample temperature applied to the reaction chamber, and a plurality of different temperatures for a TCE, wherein the plurality of different temperatures are different from each other and different from the target sample temperature and correspond to different time points, of a predetermined set of time points, of the firsts sub-step;   identify, for a first time point of the different time points for the first sub-step, a first temperature of the plurality of different temperatures using a temperature profile between the target sample temperature and the plurality of different temperatures and the first time point; and   control a temperature of the TCE at the first time point of the first sub-step to be the first temperature; wherein the TCE is in thermal communication with a reaction chamber and controls a temperature of the sample.