Rapid thermocycler system for rapid amplification of nucleic acids and related methods

Abstract

A thermocycling device and method of operating a thermocycler instrument the instrument including a sample holder, at least one thermal cycling element, and at least one first and second temperature sensors, for causing the sample holder containing the at least one sample to undergo polymerase chain reaction amplification by repeated cycling between at least a denaturation heating stage and an annealing cooling stage. The first temperature corresponding with the temperature of the sample holder is monitored using the at least one first temperature sensor, and a second temperature corresponding with the temperature external of the sample holder is monitored using the at least one second temperature sensor. Based upon the first temperature and the second temperature, the power that is delivered to the at least one thermal cycling element of the instrument is dynamically controlled.

1 . A method for operating a thermocycler instrument for amplification by polymerase chain reaction, comprising the steps of:
 a) operating the thermocycler instrument to cause a sample holder containing at least one sample to undergo a polymerase chain reaction amplification by repeated cycling between at least a denaturation heating stage and an annealing cooling stage, wherein the thermocycler instrument includes:
 (i) the sample holder which is a block with one or more bores, wherein each bore is adapted to complementarily receive a sample tube; 
 (ii) at least one thermal cycling element having at least one heat exchanger; 
 (iii) at least one first temperature sensor associated with the sample holder; and 
 (iv) at least one second temperature sensors associated with the at least one heat exchanger; 
   b) monitoring a first temperature corresponding with the temperature of the sample holder using the at least one first temperature sensor, and a second temperature corresponding with the temperature of the at least one heat exchanger external of the sample holder using the at least one second temperature sensor;   c) dynamically controlling a power that is delivered to the at least one thermal cycling element of the instrument based upon the first temperature and the second temperature: and p 1  wherein substantially real time temperature condition information obtained from the at least one first temperature sensor and the at least one second temperature sensor is employed and processed for delivering the power so it is suitable.   
     
     
         2 . The method of  claim 1 , wherein the at least one thermal cycling element includes at least one pair of thermoelectric devices which are opposed and spaced apart and the sample holder is disposed between the at least one pair of thermoelectric devices; and
 wherein the dynamically controlling step includes controlling operation of one or more parameters of at least one of the pair of thermoelectric devices.   
     
     
         3 . The method of  claim 2 , wherein the dynamically controlling step includes controlling operation of the at least one pair of thermoelectric devices and includes an alteration of the power that is delivered on the basis of a repeatedly determining an offset temperature and controlling an introduction of a respective heating pulsation during the annealing cooling stage, or a cooling pulsation during the heating stage, on the basis of the offset temperature. 
     
     
         4 . The method of  claim 2 , wherein the one or more parameters are selected from an amount of the power delivered to at least one of the pair of thermoelectric devices, a polarity of the at least one thermal cycling element, a pulse width of the power being delivered to the at least one thermal cycling element, the time that the power is delivered, or any combination thereof. 
     
     
         5 . The method of  claim 3 , wherein alteration of the power includes altering an amount of the power delivered to each of the thermoelectric devices, a polarity of each of the thermoelectric devices, a pulse width of power being delivered to each of the thermoelectric devices, the time that the power is delivered, or any combination thereof. 
     
     
         6 . The method of  claim 1 , wherein the dynamically controlling step (c) includes the steps of:
 i) receiving at least one first setpoint temperature predetermined by a user, the at least one first setpoint temperature being a maximum temperature to which the at least one sample is to be heated for polymerase chain reaction denaturation of the at least one sample;   ii) receiving at least one second setpoint temperature predetermined by the user, the at least one second setpoint temperature being a minimum temperature to which the at least one sample is to be cooled for annealing of the at least one sample;   iii) receiving at least one first hold time corresponding with an amount of time predetermined by the user during which the temperature of the sample holder is maintained generally at the at least one first setpoint temperature;   iv) receiving at least one second hold time corresponding with an amount of time predetermined by the user during which the temperature of the sample holder is maintained generally at the at least one second setpoint temperature;   v) causing the at least one thermal cycling element to heat the sample holder;   vi) receiving a first sensor signal from the at least one first temperature sensor corresponding with a temperature of the sample holder;   vii) receiving a second sensor signal from the at least one second temperature sensor corresponding with a temperature of the at least one heat exchanger and is located externally of the sample holder;   viii) determining a value of any first temperature offset amount (TOFFSET 1 ) based upon the temperature reading from the at least one first and second temperature sensors;   ix) causing heating of the sample holder until the sample holder reaches a first offset temperature that is below the first setpoint temperature by the first temperature offset amount;   x) at the time the first offset temperature is reached, causing a cooling pulsation of the at least one thermal cycling element during the heating stage for a sufficient amount of time so that the temperature arrives within about 1° C. of the first setpoint temperature and further heating of the sample holder is interrupted;   xi) causing the temperature to be maintained within about 1° C. of the first setpoint temperature for the first hold time;   xii) causing at least one thermal cycling element to cool a sample holder until the temperature of the sample holder reaches a second offset temperature that is above the second setpoint temperature by a second temperature offset (TOFFSET  2 ) amount;   xiii) receiving a temperature reading from the at least one first temperature sensor corresponding with a temperature of the sample holder.   xiv) receiving a temperature reading from the at least one second temperature sensor corresponding with a temperature of the at least one heat exchanger and is located remotely from the sample holder;   xv) determining a value of any second temperature offset amount (TOFFSET 2 ) based upon the temperature reading from the at least one first and second temperature sensors;   xvi) causing cooling of the sample holder until the sample holder reaches a second offset temperature that is above the second setpoint temperature by the second temperature offset amount;   xvii) at the time when the second offset temperature is reached, causing a heating pulsation of the at least one thermal cycling element during the cooling stage for a sufficient amount of time so that the temperature arrives within about 1° C. of the second setpoint temperature and further cooling of the sample holder is interrupted; and   xviii) causing the temperature to be maintained within about 1° C. of the second setpoint temperature for the second hold time; and   xix) repeating at least steps (v)-(xviii) for a predetermined number of cycles until the amplification desired by the user is achieved.   
     
     
         7 . The method of  claim 5 , wherein the step (c) includes the steps of:
 i) receiving at least one first setpoint temperature predetermined by a user, the at least one first setpoint temperature being a maximum temperature to which the at least one sample is to be heated for polymerase chain reaction denaturation of the at least one sample;   ii) receiving at least one second setpoint temperature predetermined by the user, the at least one second setpoint temperature being a minimum temperature to which a sample is to be cooled for annealing of the at least one sample;   iii) receiving at least one first hold time corresponding with an amount of time predetermined by the user during which the temperature of the sample holder is maintained generally at the at least one first setpoint temperature;   iv) receiving at least one second hold time corresponding with an amount of time predetermined by the user during which the temperature of the sample holder is maintained generally at the at least one second setpoint temperature;   v) causing each of the thermoelectric devices to heat the sample holder;   vi) receiving a first sensor signal from the at least one first temperature sensor corresponding with a temperature of the sample holder;   vii) receiving a second sensor signal from the at least one second temperature sensor corresponding with a temperature of the at least one heat exchanger that is located externally of the sample holder;   viii) determining a value of any first temperature offset amount (TOFFSET 1 ) based upon the temperature reading from the at least one first and second temperature sensors;   ix) causing heating of the sample holder until the sample holder reaches a first offset temperature that is below the first setpoint temperature by the first temperature offset amount;   x) at the time the first offset temperature is reached, causing a cooling pulsation of the thermoelectric devices during the heating stage for a sufficient amount of time so that the temperature arrives within about 1° C. of the first setpoint temperature and further heating of the sample holder is interrupted;   xi) causing the temperature to be maintained within about 1° C. of the first setpoint temperature for the first hold time;   xii) causing at least one thermal cycling element to cool the sample holder;   xiii) receiving a temperature reading from the at least one first temperature sensor corresponding with a temperature of the sample holder;   xiv) receiving a temperature reading from the at least one second temperature sensor corresponding with a temperature of the at least one heat exchanger and is located remotely from the sample holder;   xv) determining a value of any second temperature offset amount (TOFFSET 2 ) based upon the temperature reading from the at least one first and second temperature sensors;   xvi) causing cooling of the sample holder until the sample holder reaches a second offset temperature that is above the second setpoint temperature by the second temperature offset amount;   xvii) at the time when the second offset temperature is reached, causing a heating pulsation of the thermoelectric devices during the cooling stage for a sufficient amount of time so that the temperature arrives within about 1° C. of the second setpoint temperature and further cooling of the sample holder is interrupted;   xviii) causing the temperature to be maintained within about 1° C. of the second setpoint temperature for the second hold time; and   xix) repeating steps (v)-(xviii) for a predetermined number of cycles until the amplification desired by the user is achieved.   
     
     
         8 . The method of  claim 6 , wherein at least one of the steps of causing the temperature to be maintained includes monitoring the temperature and applying a pulse width modulated signal to the at least one thermal cycling element. 
     
     
         9 . The method of  claim 7 , wherein at least one of the steps of causing the temperature to be maintained includes monitoring the temperature and applying a pulse width modulated voltage signal to the pair of thermoelectric devices. 
     
     
         10 . A method for operating a thermocycler instrument, comprising the steps of:
 a) supplying power to a thermocycler instrument that includes;
 (i) at least two thermoelectric devices which are generally opposing and spaced apart; 
 (ii) a sample holder located between the at least two thermoelectric devices and having one or more bores to complementarily receive a sample tube; and 
 (iii) a pair of heat exchangers, each heat, exchanger associated with at least one of the at least two thermoelectric devices and projecting away from the sample holder; 
   b) applying a voltage signal having a positive polarity and a negative polarity to the at least two thermoelectric devices of the thermocycler instrument via at least one circuit;   c ) optionally, applying a voltage signal to an air mover circuit for rotating an impeller of an air mover to convectively expel air from the thermocycler instrument;   d) controlling the polarity of the voltage signal that is applied to the at least two thermoelectric devices to repeatedly alternate the operation of the thermoelectric devices between a first condition of supplying heat to the sample holder and a second condition removing heat from the sample holder;   e) obtaining a first sample holder electrical signal corresponding with a temperature within the sample holder; and   f) obtaining a second electrical signal corresponding with a temperature from at least one of the pair of heat exchangers and external of the sample holder;   g) employing the first sample holder electrical signal and the second electrical signal for determining values for controlling power delivery to be delivered to the thermoelectric devices: and   wherein substantially real time temperature condition information is employed and processed for delivering the power which is suitable to rapidly heat and cool a sample.   
     
     
         11 . The method of  claim 10 , wherein the method includes performing a series of repeated cycles that each include at least one heating stage and at least one cooling stage. 
     
     
         12 . The method of  claim 10 , wherein the method includes a heating stage that includes causing heating until the signal corresponding with a temperature within the sample holder indicates that the temperature of the sample holder has reached a first offset temperature that is within a predetermined amount below a setpoint temperature, and then causing cooling of the sample holder for a sufficient amount of time so that the temperature arrives at the setpoint with overshoot of less than about 1° C. 
     
     
         13 . The method of  claim 12 , wherein the method includes a cooling stage that includes causing cooling until the signal corresponding with a temperature within the sample holder indicates that the temperature of the sample holder has reached a second offset temperature that is within a predetermined amount above a setpoint temperature, and then causing heating of the sample holder for a sufficient amount of time so that the temperature arrives at the setpoint with undershoot of less than about 1° C. 
     
     
         14 . The method of  claim 13 , wherein in the step (f) the temperature value of the at least one of the pair of heat exchangers is employed for determining the first and second offset temperature. 
     
     
         15 . The method of  claim 14 , wherein the thermocycler is adapted to operate and is operated for heating the sample holder at a rate of at least about 8° C./second. 
     
     
         16 . The method of  claim 15 , wherein the thermocycler is adapted to operate and is operated for cooling at a rate of at least about 6° C./second. 
     
     
         17 . The method of  claim 16 , wherein the thermocycler is capable of a total runtime of less than or equal to 30 minutes for completed amplification. 
     
     
         18 . A method for controlling operation of a thermocycler instrument for amplification by polymerase chain reaction, comprising the steps of:
 a) introducing at least one biological sample into a thermocycler instrument that includes:
 i) at least one pair of thermoelectric devices which are opposed and spaced apart, each thermoelectric device having at least one heat exchanger; 
 ii) at least one sample holder which is solid metal having at least one first bore defined therein to receive at least one sample contained in a tube, and at least one second bore to receive an temperature sensor within the at least one sample holder, the at least one sample holder being disposed in thermal conducting relation with and between the at least one pair of thermoelectric devices; 
 iii) at least one first temperature sensor that is located in the at least one first bore and is adapted to monitor a first temperature of the at least one sample holder; 
 iv) at least one second temperature sensor located externally of the at least one sample holder and in a sensing relationship with at least one of the heat exchangers to monitor at least one second temperature; 
   b) receiving at least one first setpoint temperature of at least about 85° C., to which the at least one biological sample is to be heated in the sample holder for polymerase chain reaction denaturation, and at least one second setpoint temperature of below about 70° C. to which the biological sample held in the sample holder is to be cooled for annealing of the at least one biological sample;   c) maintaining a heating rate of at least about 8° C./second until a first offset temperature amount of no more than about 7.5° C. below the first setpoint temperature is reached for the sample holder;   d) when the first offset temperature is reached, pulse cooling the sample holder to slow the heating rate until the sample holder is within about 1° C. of the first setpoint temperature;   e) maintaining a cooling rate of at least about 6° C./second until a second offset temperature no more than about 7.5° C. above) the second setpoint temperature is reached for the sample holder;   f) when the second offset temperature is reached, pulse heating the sample holder to slow the cooling rate until the sample holder is within about 1° C. of the second setpoint temperature;   g) monitoring a first temperature corresponding with a temperature of the sample holder and a second temperature corresponding with a temperature of the heat exchanger;   h) adjusting an amount of time, a temperature or both at which the steps of pulse cooling, the pulse heating or both commence based upon the first temperature and the second temperature; and   i)detecting amplification in a real-time manner and repeating steps (b)-(h) for a predetermined number of cycles until the amplification desired by the user is achieved.   
     
     
         19 . The method of  claim 18 , wherein the receiving step (b) includes receiving at least one first hold time for which the at least one first setpoint temperature is desired by a user to remain substantially constant, and at least one second hold time for which the at least one second setpoint temperature is desired by a user to remain substantially constant. 
     
     
         20 . The method of  claim 19 , wherein a step of pulse width modulation is employed, while monitoring at least the temperature of the sample holder for delivering power to the thermoelectric devices for the duration of the first and second hold time in order to maintain substantially constant temperatures at each of the first and second setpoint temperatures.