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

• US 9,737,891 B2
• Filed: 05/31/2012
• Issued: 08/22/2017
• Est. Priority Date: 06/01/2011
• Status: Active Grant

• Current Assignee: Streck
• Original Assignee: Streck
• Inventors: TerMaat, Joel, Whitney, Scott E., Viljoen, Hendrik J., Kreifels, Matthew R.
• Primary Examiner(s): Chunduru, Prabha
• Application Number:
  US13/484,963
• Publication Number:
  US 20120308990A1
• Time in Patent Office: 1,909 Days
• Field of Search: None
• CPC Class Codes: B01L 2200/14 Process control and prevention of errors
• CPC Class Codes: B01L 2300/0627 Sensor or part of a sensor is integrated
• CPC Class Codes: B01L 2300/0858 Side walls
• CPC Class Codes: B01L 2300/1805 Conductive heating, heat from thermostatted solids is conducted to receptacles, e.g. heating plates, blocks
• CPC Class Codes: B01L 2300/1822 using Peltier elements
• CPC Class Codes: B01L 7/52 with provision for submitting samples to a predetermined sequence of different temperatures, e.g. for treating nucleic acid samples amplification or hybridisation processes per se C12Q1/68; controlling sequential reactions for synthesis B01J19/0046
• CPC Class Codes: C12Q 1/6844 Nucleic acid amplification reactions
• CPC Class Codes: G01N 2035/00366 Several different temperatures used

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.

Claims

1. A method for operating a thermocycler instrument for amplification by a polymerase chain reaction, comprising the steps of:

a) operating the thermocycler instrument including;

(i) a sample holder which is a block with a plurality of bores therein, wherein each bore is adapted to complementarily receive a sample tube,

(ii) a pair of thermoelectric devices opposed and spaced apart having the sample holder disposed between, each of the thermoelectric devices 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 sensor associated with the at least one beat exchanger,

for causing at least one sample in the sample tube contained in the sample holder to undergo polymerase chain reaction amplification by repealed cycling between at least a denaturation heating stage and an annealing cooling stage;

b) monitoring a first temperature corresponding with a temperature of the sample holder using the at least one first temperature sensor, and a second temperature corresponding with a temperature of the at least one heat exchanger and external of the sample holder using the at least one second temperature sensor, and 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 suitable power;

c) dynamically controlling the power that is delivered to the pair of thermoelectric devices of the thermocycler instrument based upon the first temperature and the second temperature, 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 at least one 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 the temperature of the sample holder;

(vii) receiving a second sensor signal from the at least one second temperature sensor corresponding with the 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 (TOFFSET1) 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 at least one 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 of the thermoelectric devices to cool the 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 (TOFFSET2) amount;

(xiii) receiving a temperature reading from the at least one first temperature sensor corresponding with the temperature of the sample holder;

(xiv) receiving a temperature reading from the at least one second temperature sensor corresponding with the 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 (TOFFSET2) 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 the 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 at least one 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 at least steps (v)-(xviii) for a predetermined number of cycles until the amplification desired by the user is achieved.

2. The method of claim 1,

wherein the dynamically controlling step includes controlling operation of one or more parameters of at least one of the thermoelectric devices.

3. The method of claim 1, wherein the dynamically controlling step includes controlling operation of the pair of thermoelectric devices and an alteration of the power that is delivered on a basis of repeatedly determining an offset temperature and controlling introduction of a respective heating pulsation during the annealing cooling stage, or cooling pulsation during the denaturation 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 thermoelectric devices, a polarity of at least one of the thermoelectric devices, a pulse width of the power being delivered to at least one of the thermoelectric devices, the time that the power is delivered, or any combination thereof.

5. The method of claim 3, wherein alteration of 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 the power being delivered to each of the thermoelectric devices, the time that the power is delivered, or any combination thereof.

7. The method of claim 1, 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 at least one of the thermoelectric devices.

9. The method of claim 1, wherein the thermocycler instrument is adapted to operate and is operated for heating the sample holder at a rate of at least about 8°

C./second.

10. The method of claim 1, wherein the thermocycler instrument is adapted to operate and is operated for cooling at a rate of at least about 6°

C./second.

11. The method of claim 1, wherein the thermocycler instrument is capable of a total runtime of less than or equal to 30 minutes for completed amplification.

6. A method for operating a thermocycler instrument for amplification by a polymerase chain reaction, comprising the steps of,

a) operating the thermocycler instrument including:

(i) a sample holder which is a block with a plurality of bores therein, wherein each bore is adapted to complementarily receive a sample tube,

(ii) a pair of thermoelectric devices opposed and spaced apart having the sample holder disposed between, each of the thermoelectric devices 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 sensor associated with at least one of the at least one heat exchanger,

for causing at least one sample in the sample tube contained in the sample holder to undergo polymerase chain reaction amplification by repeated cycling between at least a denaturation heating stage and an annealing cooling stage;

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 and external of the sample holder using the at least one second temperature sensor, and 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 suitable power;

c) dynamically controlling the power that is delivered to the pair of thermoelectric devices of the thermocycler instrument based upon the first temperature and the second temperature,

wherein the dynamically controlling step includes controlling operation of one or more parameters of at least one of the thermoelectric devices;

wherein the dynamically controlling step includes controlling operation of the pair of thermoelectric devices and an alteration of the power that is delivered on a basis of repeatedly determining an offset temperature and controlling introduction of a respective heating pulsation during the annealing cooling stage, or cooling pulsation during the denaturation heating stage, on the basis of the offset temperature;

wherein alteration of power includes altering an amount of 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 power is delivered, or any combination thereof; and

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 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 the temperature of the sample holder;

(vii) receiving a second sensor signal from the at least one second temperature sensor corresponding with the 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 (TOFFSET1) based upon a 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 of the thermoelectric devices to cool the sample holder;

(xiii) receiving a temperature reading from the at least one first temperature sensor corresponding with if the temperature of the sample holder;

(xiv) receiving a temperature reading from the at least one second temperature sensor corresponding with the temperature of the at least one heat exchanger that is located remotely from the sample holder;

(xv) determining a value of any second temperature offset amount (TOFFSET2) 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 voltage signal to the thermoelectric devices.

12. 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 and at least one fluorescent agent into a thermocycler instrument that includes;

(i) at least one pair of thermoelectric devices which are opposing and spaced apart and have one or more associated heat exchangers;

(ii) at least one sample holder which is a substantially solid metal block and is disposed in thermal conducting relation with and between the at least one pair of thermoelectric devices, wherein the sample holder includes;

a plurality of bores defined therein and each bore is adapted to receive a sample contained in a tube, and

at least one sensor bore to receive a temperature sensor within the sample holder;

(iii) at least one first temperature sensor that is located in the sensor 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 sample holder and in a sensing relationship with at least one of the one or more heat exchangers to monitor at least one second temperature;

(iv) a detector;

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 by 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 with the at least one first temperature sensor and a second temperature corresponding with a temperature of at least one of the one or more heat exchangers with the at least one second temperature sensor;

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 mariner and repeating steps (b)-(h) for a predetermined number of cycles until the amplification desired by a user is achieved.

13. The method of claim 12, 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 the user to remain substantially constant, and at least one second hold time for which the at least one second setpoint temperature is desired by the user to remain substantially constant.

14. The method of claim 13, wherein a step of pulse width modulation is employed, while monitoring at least the temperature of the sample holder for delivering power to the pair of 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.