U.S. patent application number 14/521232 was filed with the patent office on 2015-02-12 for adaptive thermal block temperature control method and system.
The applicant listed for this patent is APPLIED BIOSYSTEMS, LLC. Invention is credited to Chee Wee Ching, Chee Kiong Lim.
Application Number | 20150044727 14/521232 |
Document ID | / |
Family ID | 39766500 |
Filed Date | 2015-02-12 |
United States Patent
Application |
20150044727 |
Kind Code |
A1 |
Lim; Chee Kiong ; et
al. |
February 12, 2015 |
Adaptive Thermal Block Temperature Control Method and System
Abstract
Aspects of the present teachings describe a method and apparatus
for automatically controlling a block temperature to reduce
undershooting and overshooting of the temperatures of a sample
contained in the block and participating in a polymerase chain
reaction (PCR). The adaptive thermal block temperature control
begins when a sample temperature enters a sample window region
between a preliminary setpoint temperature and a target setpoint
temperature for the sample. Based on thermodynamic behavior of the
sample and the predetermined phase of PCR, predicting a time period
measured subsequent to the preliminary setpoint temperature when
the sample will reach the target setpoint suitable for the
predetermined phase of PCR. During this time period, varying the
block temperature ramp rate with a series of cooling and heating
changes to ensure the block temperature reaches the target setpoint
temperature at approximately the same time as the sample reaches
the same. Synchronizing the block temperature and sample
temperature to the target setpoint temperature reduces
undershooting and overshooting of the sample temperature and
increases the speed and efficiency of the overall PCR process as it
relates to the thermal cycling operations.
Inventors: |
Lim; Chee Kiong; (Singapore,
SG) ; Ching; Chee Wee; (Singapore, SG) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
APPLIED BIOSYSTEMS, LLC |
Carlsbad |
CA |
US |
|
|
Family ID: |
39766500 |
Appl. No.: |
14/521232 |
Filed: |
October 22, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
12053416 |
Mar 21, 2008 |
8871470 |
|
|
14521232 |
|
|
|
|
60896087 |
Mar 21, 2007 |
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Current U.S.
Class: |
435/91.2 ;
435/286.1 |
Current CPC
Class: |
C12P 19/34 20130101;
B01L 2200/147 20130101; B01L 2300/1827 20130101; G05D 23/32
20130101; B01J 19/0046 20130101; B01L 7/52 20130101 |
Class at
Publication: |
435/91.2 ;
435/286.1 |
International
Class: |
B01J 19/00 20060101
B01J019/00; C12P 19/34 20060101 C12P019/34 |
Claims
1. A computer implemented method of controlling a thermal cycler
for use in PCR, comprising; determining a current temperature ramp
rate for a sample being processed in a thermal block; predicting an
expected time interval for the sample to reach a target setpoint
temperature based upon the current temperature ramp rate for the
sample; operating the thermal block at a target block ramp rate
according to the predicted time interval for the sample to reach
the target setpoint temperature; repeating determination of the
current temperature ramp rate for the sample as the thermal
protocol for PCR is performed in real-time; determining whether the
predicted time interval for sample to reach target setpoint
temperature has changed based on the current temperature ramp rate;
and modifying the predicted time interval and the target block ramp
rate to ensure target block and sample both reach target setpoint
temperature at approximately same time.
2. The computer implemented method of claim 1 wherein repeating the
determination of the current temperature ramp rate occurs at a
frequency corresponding to control loop time interval.
3. The computer implemented method of claim 1 wherein the control
loop time interval is approximately 20 times per second.
4. The computer implemented method of claim 1 wherein modifying the
predicted time interval occurs during an adaptive window for
adjusting the target block ramp rate of the thermal block.
5. The computer implemented method of claim 4 wherein the adaptive
window designates approximately where the sample temperature is
predicted to reach the target setpoint temperature and wherein the
target block ramp rate is modified so achieve substantially the
same sample temperature and block temperature within the range of
the adaptive window.
6. An apparatus for controlled automated performance of polymerase
chain reactions, the apparatus comprising: at least one sample
comprising a PCR mixture to be amplified and contained in a sample
vessel whose temperature is varied by association with a thermal
block of variable temperature; and a control module implementing a
PCR thermal protocol configured to vary the temperature of the
thermal block and further configured to perform the steps of:
determining a current temperature ramp rate for the sample;
predicting an expected time interval for the sample to reach a
target setpoint temperature based upon the current temperature ramp
rate for the sample; operating the thermal block at a thermal block
ramp rate according to the predicted time interval for the sample
to reach the target setpoint temperature; repeating determination
of the current temperature ramp rate for the sample as the thermal
protocol for PCR is performed; determining whether the predicted
time interval for sample to reach target setpoint temperature has
changed based on the current temperature ramp rate; and modifying
the predicted time interval and the thermal block ramp rate such
that the thermal block and sample reach target setpoint temperature
at approximately same time.
7. The apparatus of claim 6 wherein repeating the determination of
the current temperature ramp rate occurs at a frequency
corresponding to control loop time interval.
8. The apparatus of claim 7 wherein the control loop time interval
is approximately 20 times per second.
9. The apparatus of claim 6 wherein modifying the predicted time
interval occurs during an adaptive window for adjusting the thermal
block ramp rate of the thermal block.
10. The apparatus of claim 9 wherein the adaptive window designates
approximately where the sample temperature is predicted to reach
the target setpoint temperature and wherein the target block ramp
rate is modified so achieve substantially the same sample
temperature and block temperature within the range of the adaptive
window.
11. A method for polymerase chain reaction (PCR) temperature
control, the method comprising the steps of; determining a current
temperature ramp rate for a sample processed in a thermal block;
predicting an expected time interval for the sample to reach a
target setpoint temperature based upon the current temperature ramp
rate for the sample; operating the thermal block at a target block
ramp rate according to the predicted time interval for the sample
to reach the target setpoint temperature; repeating determination
of the current temperature ramp rate for the sample as the thermal
protocol for PCR is performed; determining whether the predicted
time interval for sample to reach target setpoint temperature has
changed based on the current temperature ramp rate; and modifying
the predicted time interval and the target block ramp rate to
ensure target block and sample reach target setpoint temperature at
approximately same time.
12. The method of claim 11 wherein repeating the determination of
the current temperature ramp rate occurs at a frequency
corresponding to control loop time interval.
13. The method of claim 12 wherein the control loop time interval
is approximately 20 times per second.
14. The method of claim 11 wherein modifying the predicted time
interval occurs during an adaptive window for adjusting the target
block ramp rate of the thermal block.
15. The method of claim 14 wherein the adaptive window designates
approximately where the sample temperature is predicted to reach
the target setpoint temperature and wherein the target block ramp
rate is modified so achieve substantially the same sample
temperature and block temperature within the range of the adaptive
window.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional of U.S. application Ser.
No. 12/053,416 filed Mar. 21, 2008, which claims the benefit of
U.S. Provisional Application No. 60/896,087 filed Mar. 21, 2007,
both of which are incorporated herein by reference.
FIELD
[0002] The present teachings relate to the field of instruments for
performing polymerase chain reaction. More particularly, the
present teachings pertain to systems and methods for temperature
control in instruments capable of performing polymerase chain
reaction
INTRODUCTION
[0003] Polymerase Chain Reaction (PCR) has proven a phenomenally
successful technology for genetic analysis. A key aspect of PCR is
the concept of thermocycling: alternating steps of melting a
nucleic acid template, annealing primers to the resulting single
strands, and extending those primers to make new copies of double
stranded nucleic acid. In thermocycling, a PCR reaction mixture may
be repeatedly cycled from high temperatures for melting the DNA, to
lower temperatures for primer annealing and extension.
[0004] In a typical PCR reaction, the reaction mixture is desirably
transitioned and maintained accurately at various temperatures for
prescribed time periods with temperature cycling frequently
repeated many times. Generally, it is desirable to change the
sample temperature to the next temperature in the cycle rapidly for
several reasons. First, the chemical reaction may have an optimum
temperature for each of its stages. Thus, less time spent at
nonoptimum temperatures may improve the result product. Another
reason is that a minimum time for holding the reaction mixture at
each incubation temperature may be desired after each incubation
temperature is reached. These minimum incubation times may
establish the "floor" or minimum time it takes to complete a cycle.
Any time transitioning between sample incubation temperatures is
time which is added to this minimum cycle time. Since the number of
cycles is often fairly large, this additional time lengthens the
total time needed to complete the amplification. Another important
consideration is achieving each desired sample temperature with
minimal under and/or overshooting which may adversely affect the
resultant product or increase the overall reaction time.
BRIEF DESCRIPTION OF THE DRAWINGS
[0005] The skilled artisan will understand that the drawings,
described below, are for illustration purposes only. The drawings
are not intended to limit the scope of the present teachings in any
way.
[0006] FIG. 1 is a schematic illustrating a block diagram of the
major system components of one embodiment of a computer directed
instrument for performing PCR in accordance with various
implementations of the present teachings;
[0007] FIG. 2 is a schematic illustrating an alternative
peltier-based thermocycler component in accordance with various
implementations of the present teachings;
[0008] FIG. 2A is a schematic illustrating another view of the
alternative peltier-based thermocycler component in accordance with
various implementations of the present teachings;
[0009] FIG. 3A is a schematic illustrating one embodiment of a
power control concept for a film heater in accordance with various
implementations of the present teachings;
[0010] FIG. 3B is a schematic illustrating a time versus
temperature plot of a typical PCR protocol.
[0011] FIG. 3C illustrates two local regions side by side for a
design of a sample block in accordance with aspects of the present
teachings.
[0012] FIG. 4 is a schematic illustrating three separately
controlled zones within a film heater layer in accordance with
various implementations of the present teachings;
[0013] FIG. 5A is a schematic illustrating an example of operating
a sample block at ramp rates that cause the sample temperature to
either overshoot or undershoot setpoint targets.
[0014] FIG. 5B is a schematic illustrating an example block
temperature that operates with a rapid ramp rate to achieve a
sample temperature timing specified in a thermal protocol
[0015] FIG. 6 is a flowchart diagram of the operations for
performing an adaptive thermal block control in accordance with
aspects of the present teachings.
SUMMARY
[0016] In various embodiments the present teachings describe a
computer implemented method of controlling a thermal cycler for use
in PCR. The method further comprising: determining a current
temperature ramp rate for a sample being processed in a thermal
block; predicting an expected time interval for the sample to reach
a target setpoint temperature based upon the current temperature
ramp rate for the sample; operating the thermal block at a target
block ramp rate according to the predicted time interval for the
sample to reach the target setpoint temperature; repeating
determination of the current temperature ramp rate for the sample
as the thermal protocol for PCR is performed in real-time;
determining whether the predicted time interval for sample to reach
target setpoint temperature has changed based on the current
temperature ramp rate; and modifying the predicted time interval
and the target block ramp rate to ensure target block and sample
both reach target setpoint temperature at approximately same
time.
[0017] In another embodiment the present teachings describe an
apparatus for controlled automated performance of polymerase chain
reactions. The apparatus further comprising: at least one sample
comprising a PCR mixture to be amplified and contained in a sample
vessel whose temperature is varied by association with a thermal
block of variable temperature; and a control module implementing a
PCR thermal protocol configured to vary the temperature of the
thermal block and further configured to perform the steps of:
determining a current temperature ramp rate for the sample;
predicting an expected time interval for the sample to reach a
target setpoint temperature based upon the current temperature ramp
rate for the sample; operating the thermal block at a thermal block
ramp rate according to the predicted time interval for the sample
to reach the target setpoint temperature; repeating determination
of the current temperature ramp rate for the sample as the thermal
protocol for PCR is performed; determining whether the predicted
time interval for sample to reach target setpoint temperature has
changed based on the current temperature ramp rate; and modifying
the predicted time interval and the thermal block ramp rate such
that the thermal block and sample reach target setpoint temperature
at approximately same time.
[0018] In yet other embodiments, the present teachings describe a
method for polymerase chain reaction (PCR) temperature control. The
method comprising the steps of; determining a current temperature
ramp rate for a sample processed in a thermal block; predicting an
expected time interval for the sample to reach a target setpoint
temperature based upon the current temperature ramp rate for the
sample; operating the thermal block at a target block ramp rate
according to the predicted time interval for the sample to reach
the target setpoint temperature; repeating determination of the
current temperature ramp rate for the sample as the thermal
protocol for PCR is performed; determining whether the predicted
time interval for sample to reach target setpoint temperature has
changed based on the current temperature ramp rate; and modifying
the predicted time interval and the target block ramp rate to
ensure target block and sample reach target setpoint temperature at
approximately same time.
[0019] These and other features of the present teachings are set
forth herein.
DESCRIPTION
[0020] The present teachings provide improved methods for
temperature control in PCR processes. Details of the polymerase
chain reaction process, the temperature cycling and reaction
conditions used in PCR as well as the various reagents and enzymes
used to perform the reaction are described in U.S. Pat. Nos.
4,683,202, 4,683,195, EPO Publication 258,017 and U.S. Pat. No.
4,889,818, which are hereby incorporated by reference. Details of
instruments for use in PCR are described in U.S. Pat. Nos.
5,475,610 and 7,133,726 assigned to the assignee of the present
invention and which are incorporated herein by reference.
[0021] In various PCR instruments, the reaction mixture may be
stored or contained in a tube, well, through-hole or other fluid
containment region provided by a substrate or vessel. A typical
sample volume may be between approximately 10 nanoliters and 1000
microliters although greater or lesser amounts of reaction mixture
may be readily amplified. Typically, such instruments may be
configured to simultaneously amplify multiple sample reaction
mixtures accomplished by heat transfer to and from an associated
heat transfer block (for example, a metal or metal alloy sample
block). In various embodiments, the PCR process is performed by
controlling the temperature of the heat transfer block according to
prescribed temperatures and times specified by the user in a PCR
protocol file.
[0022] A computer and associated electronics controls the
temperature of the heat transfer block in accordance with the user
supplied data in the PCR protocol file defining the times,
temperatures and number of cycles, etc. As the heat transfer block
changes temperature, the samples follow with similar changes in
temperature. However, one challenge in heating each sample is to
maintain a consistent temperature between all samples while at the
sample time raising and lowering sample temperatures accurately.
Prior art PCR instruments typically possess a degree of error in
sample temperatures generated by nonuniformity of temperature from
place to place within the heat transfer block as well as suffering
from a lack accuracy when raising and lowering the temperature of
all samples in comparison to a desired temperature profile.
[0023] In one aspect, delays in transferring heat between the
sample block and the sample creates deviations from desired
temperature ramping profiles. To change the sample temperature to a
setpoint level, the sample block is generally configured to
exchange the appropriate amount of heat with the samples in the
sample block. Allowing too much heat transfer from the sample block
can cause a sample to either overshoot or undershoot the setpoint
level depending on the temperature ramp rate for the sample and the
sample block heat exchange characteristics.
[0024] Undershooting and/or overshooting temperatures may also
introduce inaccuracies in a particular protocol. For example, if
the sample temperature overshoots/undershoots the setpoint level
then the particular PCR protocol may not perform as designed,
amplify less efficiently or fail to work at all. To improve the PCR
process and help aid in successful and efficient reaction
amplification, it is desirable to bring the sample temperature to
the various setpoint levels without overshooting/undershooting the
sample temperature. This is particular important in performing
"quantitative" PCR where time delays and temperature errors need to
be minimized. Achieving this goal can be increasingly difficult
when the size of the heat transfer block used to heat and cool the
samples is relatively large. In one respect, the relatively large
thermal mass of the block may present difficulties in transitioning
the block temperature up and down in the operating range with great
rapidity. Additionally, the block may be associated with various
external devices such as manifolds for supply and withdrawal of
cooling liquid, block support attachment points, peltier devices,
heat sinks and associated other peripheral equipment which create
the potential for temperature gradients to exist across the block
which exceed tolerable limits.
[0025] Referring to FIG. 1 there is a block diagram of the major
system components of one embodiment of a computer directed
instrument for performing PCR according to the teachings of the
present teachings. Sample mixtures including the DNA or RNA to be
amplified are placed in the temperature-programmed sample block 12
and may be covered by heated cover 14.
[0026] A user supplies data defining time and temperature
parameters of the desired PCR protocol via a terminal 16 including
a keyboard and display. The keyboard and display are coupled via
bus 18 to a control computer 20 (hereafter sometimes referred to as
a central processing unit or CPU). This central processing unit 20
includes memory which stores the control program, data defining the
desired PCR protocol and calibration constants. The control program
causes the CPU 20 to control temperature cycling of the sample
block 12 and implements a user interface which provides certain
displays to the user and which receives data entered by the user
via the keyboard of the terminal 16.
[0027] In one implementation, the central processing unit 20 is
custom designed to facilitate improved performance and control over
temperature cycling of the sample block 12. In alternative
embodiments, the central processing unit 20 and associated
peripheral electronics to control the various heaters and other
electro-mechanical systems of the instrument and read various
sensors could be any general purpose computer such as a suitably
programmed personal computer or microcomputer.
[0028] The samples 10 are contained in vessels which are seated in
or in proximity with the sample block 12 and may be thermally
isolated from the ambient air by a heated cover 14. The heated
cover 14 may serve, among other things, to reduce undesired heat
transfers to and from the sample mixture by evaporation,
condensation and refluxing inside the sample tubes. It may also
reduce the chance of cross contamination by keeping the insides of
the caps dry thereby preventing aerosol formation when the tubes
are uncapped.
[0029] The central processing unit 20 may include appropriate
electronics to sense the temperature of the heated cover 14 and
control electric resistance heaters therein to maintain the cover
14 at a predetermined temperature. Sensing of the temperature of
the heated cover 14 and control of the resistance heaters therein
may be accomplished via a temperature sensor (not shown) and bus
22.
[0030] In one exemplary embodiment a coolant control system 24
continuously circulates a chilled liquid coolant through bias
cooling channels (not shown) in the sample block 12 via input tubes
26 and output tube 28. The coolant control system 24 also controls
fluid flow through higher volume ramp cooling fluid flow paths (not
shown) in the sample block 12. The ramp cooling channels are used
to rapidly change the temperature of the sample block 12 by pumping
large volumes of chilled liquid coolant through the block at a
relatively high flow rate. Ramp cooling liquid coolant enters the
sample block 12 through tube 30 and exits the sample block through
tube 32.
[0031] The liquid coolant used to chill the sample block 12 may
comprise a mixture of water and ethylene glycol. The liquid coolant
may be chilled by a heat exchanger 34 that receives liquid coolant
with heat extracted from the sample block 12 via input tube 36. The
heat exchanger 34 receives compressed liquid Freon refrigerant via
input tube 38 from a refrigeration unit 40. This refrigeration unit
40 includes a compressor (not shown), a fan 42 and a fin tube heat
radiator 44. The refrigeration unit 40 compresses Freon gas
received from the heat exchanger 34 via tube 46. The gaseous Freon
is cooled and condensed to a liquid in the fin tube condenser 44.
The pressure of the liquid Freon is maintained above its vapor
pressure in the fin tube condenser 44 by a flow restrictor
capillary tube 47. The output of this capillary tube 47 is coupled
to the input of the heat exchanger 34 via input tube 38. In the
heat exchanger 34, the pressure of the Freon is allowed to drop
below the Freon vapor pressure, and the Freon expands.
[0032] During the expansion process, heat is absorbed from the
warmed liquid coolant circulating in the heat exchanger 34 and this
heat is transferred to the Freon thereby causing the Freon to boil.
The warmed Freon is then extracted from the heat exchanger 34 via
tube 46 and is compressed and again circulated through the fin tube
condenser 44. The fan 42 blows air through the fin tube condenser
44 to cause heat in the freon from tube 46 to be exchanged with the
ambient air. As symbolized by arrows 48. In one embodiment, the
refrigeration unit 40 should be capable of extracting 400 watts of
heat at 30.degree C. and 100 watts of heat at 10 degree C. from the
liquid coolant to support the rapid temperature cycling as needed
in various aspects of the present teachings.
[0033] After exchanging its heat with the Freon, the liquid coolant
exits the heat exchanger 34 via tube 50 and reenters the coolant
control system where it is gated as needed to the sample block
during rapid cooling portions of the PCR cycle defined by data
entered by the user via terminal 16.
[0034] An alternative sample heating apparatus may include a
Peltier based thermoelectric device such as those described in
commonly assigned U.S. Pat. No. 7,133,726. Heat-pumping into and
out of the samples is accomplished by using a Peltier
thermoelectric component that may be constructed of pellets of
n-type and p-type bismuth telluride connected alternately in
series. The interconnections between the pellets may be made with
copper which is bonded to a substrate, usually a ceramic (typically
alumina).
[0035] The amount of heat-pumping desired is dependent on the
thermal load and the ramp rate, that is, the rate at which the
temperature is required to change. Factors such as the composition
and configuration of the sample block, thermoelectric devices,
heatsink, fan and the thermal interface media between the
thermoelectric devices and both the heatsink and the sample block
may also affect the heat-pumping parameters. In these devices
samples may be heated by an apparatus depicted in FIGS. 2 and 2A
reflecting a typical Peltier thermal electric device 60. The device
is composed of bismuth telluride pellets 62, sandwiched between two
alumna layers 64. The pellets are electrically connected by solder
joints 66 to copper traces 68 plated onto the alumina layers. One
alumina layer has an extension 69 to facilitate electrical
connections. The thickness of the extended areas may be reduced to
decrease the thermal load of the device.
[0036] Generally PCR reaction temperatures occur above ambient for
example in the range 30 to 104.degree. C. In the most cases the
block is heated or cooled between at least two above ambient
temperatures where the flow of heat due to conduction is from the
block to the heat sink. In one aspect, system cycle time may be
optimized for a given block configuration to achieve a desired
balance between the boost to the ramp rate when cooling provided by
the conduction, against the boost provided to the heating ramp rate
by the Joule effect of resistance heating.
[0037] FIG. 3B exemplifies a typical PCR cycle with a denaturation
incubation 170 done at a temperature near 94 degree C., a
hybridization incubation 172 done at a temperature near room
temperature (25 degree C. to 37 degree C.) and an extension
incubation 174 done at a temperature near 50 degree C. These
temperatures are substantially different, and, therefore it is
desirable to have equipment and methods of moving the temperature
of the reaction mixture of all the samples rapidly from one
temperature to another.
[0038] In operation using the exemplary thermalcycler configuration
shown in FIG. 1, CPU 20 controls multi-zone heater 156 via bus 52.
The temperature of multi-zone heater 156 can be controlled to raise
the temperature of the sample block 12 rapidly to higher incubation
temperatures from lower incubation temperatures. It is also capable
of compensating for bias cooling and correcting temperature errors
in the upward direction during temperature tracking and during
incubations. In alternative embodiments, bias cooling may be
eliminated or may be supplied by other means such as by the use of
a cooling fan and cooling fins formed in the metal of the sample
block, peltier junctions or constantly circulating tap water.
[0039] For the liquid cooled apparatuses CPU 20 controls the
temperature of the sample block 12 by sensing the temperature of
the metal of the sample block via temperature sensor 21 and bus 52
in FIG. 1 and by sensing the temperature of the circulating coolant
liquid via bus 54 and a temperature sensor in the coolant control
system. The CPU also senses the internal ambient air temperature
within the housing of the system via an ambient air temperature
sensor 56 in FIG. 1. Further, the CPU 20 senses the line voltage
for the input power on line 58 via a sensor symbolized at 63.
[0040] A control program uses these items of data together with
items of data entered by the user to define the desired PCR
protocol such as target temperatures and times for incubations.
This control program calculates the amount of power to apply to the
various zones of the multi-zone sample block film heater 156 via
the bus 52 and generates a coolant control signal to open or close
the solenoid operated valve 55 in the coolant control system 24 via
bus 54 causing the temperature of the sample block to follow the
PCR protocol defined by data entered by the user. As will be
appreciated by one of skill in the art, the temperature control
provided by the CPU 20 may be adapted for use with other
heating/cooling configurations such as solid state/peltier-based
systems or other components which heat and cool the sample block to
achieve the desired temperature profiles for sample
thermocycling.
[0041] Irrespective of the manner in which the sample is heated and
cooled, it is particularly important to perform PCR amplification
with a high degree of temperature control and precision.
Accordingly, it is important to maintain precise control over
sample mixture temperature as between various ones of a
multiplicity of different samples. For example, if all the samples
are not precisely controlled to have the proper annealing
temperature for the extension incubation certain forms of DNA may
not extend properly. This happens because the primers used in the
extension process may anneal to the wrong DNA template if the
temperature is too low. If the annealing temperature is too high,
the primers may not anneal to the target DNA at all.
[0042] FIG. 3A illustrates one exemplary embodiment of a power
control concept that may be used in connection with the film heater
156, peltier-based thermal transfer approach and other thermocycler
designs or configurations. FIG. 3A diagrams an exemplary voltage
waveform for a supply line voltage. Rectification to eliminate the
negative half cycle 162 may occur and in certain embodiments only
positive half cycles may remain of which half cycle 164 is typical.
The CPU 20 and its associated peripheral electronic circuitry may
then control the portion of each half cycle which is applied to the
various zones of the film heater 156 by selecting a portion of each
half cycle to apply according to a power level computed for each
zone based upon equations given below for each zone. That is, the
dividing line 166 is moved forward or backward along the time axis
to control the amount of power to the film heater based upon a
number of factors which are related in a special equation for each
zone. The cross-hatched area under the positive half cycle 164
represents the amount of power applied to the film heater 156 for
the illustrated position of the dividing line 166. As the dividing
line 166 is moved to the right, more power is applied to the film
heater, and the sample block 12 gets hotter. As the dividing line
is moved to the left along the time axis, the cross-hatched area
becomes smaller and less power is applied to the film heater.
[0043] Referring to FIG. 3B, there is shown a time versus
temperature plot of a typical PCR protocol. Large downward changes
in block temperature are accomplished by cooling the sample block
while monitoring the sample block temperature by the temperature
sensor 21 in FIG. 1. Typically these rapid downward temperature
changes are carried out during the ramp following the denaturation
incubation 170 to the temperature of hybridization incubation 172.
Typically, the user must specify the protocol by defining the
temperatures and times in one fashion or another so as to describe
to the CPU 20 the positions on the temperature/time plane of the
checkpoints symbolized by the circled intersections between the
ramp legs and the incubation legs. Generally, the incubation legs
are marked with reference numerals 170, 172 and 174 and the ramps
are marked with reference numerals 176, 178 and 180.
[0044] Generally the incubation intervals are conducted at a single
temperature, but in alternative embodiments, they may be stepped or
continuously ramped to different temperatures within a range of
temperatures which is acceptable for performing the particular
portion of the PCR cycle involved. That is, the denaturation
incubation 170 need not be carried out at one temperature as shown
in FIG. 3B, but may be carried out at any of a plurality of
different temperatures within the range of temperatures acceptable
for denaturation. In some embodiments, the user may specify the
length of the ramp segments 176, 178 and 180. In other embodiments,
the user may only specify the temperature or temperatures and
duration of each incubation interval, and the instrument will then
move the temperature of the sample block as rapidly as possible
between incubation temperatures upon the completion of one
incubation and the start of another. In the preferred embodiment,
the user can also have temperatures and/or incubation times which
are different for each cycle or which automatically increment on
every cycle.
[0045] In one exemplary embodiment, the amount heat added to or
removed is estimated from the block, the CPU 20 measures the block
temperature using temperature sensor 21 in FIG. 1 and measures the
coolant temperature by way of temperature sensor coupled to bus 54
in FIG. 1. In addition, CPU 20 uses additional sensors to measure
ambient air temperature and the power line voltage, which controls
the power applied to the film heaters on bus 52. The thermal
conductance from the sample block to ambient air and from the
sample block to the coolant are known to the CPU 20 as a result of
measurements made during an initialization process to set control
parameters of the system.
[0046] For good temperature uniformity of the sample population,
the block, at constant temperature, should have little or no net
heat flow in or out. However, temperature gradients can occur
within the sample block arising from local flows of heat from hot
spots to cold spots which have zero net heat transfer relative to
the block borders. For instance, a slab of material which is heated
at one end and cooled at the other is at a constant average
temperature if the net heat flow into the block is zero. However,
in this situation a significant temperature nonuniformity, e.g., a
temperature gradient, can be established within the slab due to the
flow of heat from the hot edge to the cold edge. When heating and
cooling of the edges of the block are stopped, the flow of heat
from the hot edge to the cold edge eventually dissipates this
temperature gradient and the block reaches a uniform temperature
throughout which is the average between the hot temperature and
cool temperature at the beginning of heat flow.
[0047] Practically speaking, it is not always practical to control
the temperature of a sample block without some heat flow in and
out. The cold bias control cooling requires some heat flow in from
the strip heaters to balance the heat removed by the coolant
flowing through the bias cooling channels to maintain the block
temperature at a stable value. The key to a uniform sample block
temperature under these conditions is a geometry which has "local
balance" and "local symmetry" of heat sources and heat sinks both
statically and dynamically, and which is arranged such that any
heat flow from hot spots to cold spots occurs only over a short
distance.
[0048] Stated briefly, the concept of "static local balance" means
that in a block at constant temperature where the total heat input
equals the total heat output, the heat sources and heat sinks are
arranged such that within a distinct local region, all heat sources
are completely balanced by heat sinks in terms of heat flows in and
heat flows out of the block. Therefore, each local region, if
isolated, would be maintained at a constant temperature.
[0049] The concept of "static local symmetry" means that, within a
local region and for a constant temperature, the center of mass of
heat sources is coincident with the center of mass of heat sinks.
If this were not the case, within each local region, a temperature
gradient across each local region can exist which can add to a
temperature gradient in an adjacent local region thereby causing a
gradient across the sample block which is twice as large as the
size of a single local region because of lack of local symmetry
even though local balance within each local region exists. The
concepts of local balance and local symmetry are important to the
achievement of a static temperature balance where the temperature
of the sample block is being maintained at a constant level during,
for example, an incubation interval.
[0050] For the dynamic case where rapid temperature changes in the
sample block are occurring, the thermal mass, or heat capacity of
each local region becomes important. This is because the amount of
heat that must flow into each local region to change its
temperature is proportional to the thermal mass of that region.
[0051] Therefore, the concept of static local balance can be
expanded to the dynamic case by requiring that if a local region
includes x percent of the total dynamic heat source and heat sink,
it must also include x percent of the thermal mass for "dynamic
local balance" to exist. Likewise, "dynamic local symmetry"
requires that the center of mass of heat capacity be coincident
with the center of mass of dynamic heat sources and sinks. What
this means in simple terms is that the thermal mass of the sample
block is the metal thereof, and the machining of the sample block
must be symmetrical and balanced such that the total mass of metal
within each local zone is the same. Further, the center of mass of
the metal in each local zone should be coincident with the center
of mass of the dynamic heat sources and sinks. Thus, the center of
mass of the multi-zone heater 156, e.g., its geometric center, and
the geometric center of the bias and ramp cooling channels must
coincide.
[0052] FIG. 3C illustrates two local regions side by side for the
design of the sample block 12 in accordance with aspects of the
present teachings. In FIG. 3C, the boundaries of two local regions,
200 and 202, are marked by dashed lines 204, 206 and 208. FIG. 3C
shows that each local region which is not in the guard band is
comprised of: two columns of sample wells; a portion of the foil
heater 156 which turns out to be 1/8th of the total area of the
heater; one ramp cooling channel such as ramp cooling channels 210
and 212; and, one bias cooling channel. To preserve local symmetry,
each local region is centered on its ramp cooling channel and has
one-half of a bias cooling channel at each boundary.
[0053] For example, local region 200 has a center over the ramp
cooling channel 210 and bias cooling channels 214 and 216 are
dissected by the local region boundaries 204 and 206, respectively.
Thus the center of mass of the ramp cooling channel (the middle
thereof), coincides (horizontally) with the center of mass of the
bias cooling channels (the center of the local region) and with the
center of mass of the film heater portion coupled to each local
region. Static local balance will exist in each local region when
the CPU 20 is driving the film heater 156 to input an amount of
heat energy that is equal to the amount of heat energy that is
being removed by the ramp cooling and bias cooling channels.
[0054] Dynamic local balance for each local region exists because
each local region in the center portion of the block where the 96
sample mixtures reside contains approximately 1/8th the total
thermal mass of the entire sample block, contains 1/8th of the
total number of ramp cooling channels and contains 1/8th of the
total number of bias cooling channels. Dynamic local symmetry
exists for each local region, because the center of mass of the
metal of each local region is horizontally coincident with the
center of film heater portion underlying the local region; the
center of the ramp cooling channel; and, the center of mass of the
two half bias cooling channels. By virtue of these physical
properties characterized as static and dynamic local balance and
local symmetry, the sample block heats and cools all samples in the
population uniformly.
[0055] Referring to FIG. 4A, there are shown three separately
controlled zones within the film heater layer 156. These separately
controlled zones include edge heater zones which are situated under
the guard bands at the exposed edges of the sample block 12 which
are coupled to the support bracket 148. There are also separately
controlled manifold heater zones situated under the guard bands for
the edges 228 and 230 which are attached to the coolant manifolds.
Finally, there is a central heater zone that underlies the sample
wells. The power applied to each of these zones is separately
controlled by the CPU 20 and the control software.
[0056] The film heater 156 is composed of a pattern of electrical
conductors formed by etching a thin sheet of metal alloy such as
Inconel.TM.. The metal alloy selected should have high electrical
resistance and good resistance to heat. The pattern of conductors
so etched is bonded between thin sheets of an electrically
insulating polymeric material such as Kapton.TM.. Whatever material
is used to insulate the electrical resistance heating element, the
material must be resistant to high temperatures, have a high
dielectric strength and good mechanical stability.
[0057] The central zone 254 of the film heater has approximately
the same dimensions as the central portion of the sample block
inside the guard bands. Central region 254 delivers a uniform power
density to the sample well area. Edge heater regions 256 and 258
are about as wide as the edge guard bands but are not quite as
long. Manifold heater regions 260 and 262 underlie the guard bands
for edges.
[0058] The manifold heater zones 260 and 262 are electrically
connected together to form one separately controllable heater zone.
In addition, the edge heater sections 256 and 258 are electrically
coupled together to form a second separately controllable heater
zone. The third separately controllable heater zone is the central
section 254. Each of these three separately controllable heater
zones has separate electrical leads, and each zone is controlled by
a separate control algorithm which may be run on separate
microprocessors or a shared CPU as is done in various
embodiments.
[0059] The edge heater zones 256 and 258 are driven to compensate
for heat lost to the support brackets. This heat loss is
proportional to the temperature difference between the sample block
12 and the ambient air surrounding it. The edge heater zones 256
and 258 also compensate for the excess loss of heat from the sample
block to the full bias cooling channels at each edge of the block.
This heat loss is proportional to the temperature difference
between the sample block 12 and the coolant flowing through these
bias cooling channels.
[0060] The manifold heater sections 260 and 262 are also driven so
as to compensate for heat lost to the plastic coolant manifolds 266
and 268 in FIG. 4A which are attached to the edges of the sample
block 12. The power for the manifold heater sections 260 and 262
compensates for heat loss which is proportional mainly to the
temperature difference between the sample block and the coolant,
and to a lesser degree, between the sample block and the ambient
air.
[0061] The control algorithm run by CPU 20 of FIG. 1 senses the
temperature of the sample block via temperature sensor 21 in FIG. 1
and FIG. 9 and bus 52 in FIG. 1. This temperature is differentiated
to derive the rate of change of temperature of the sample block 12.
The CPU then measures the temperature of the ambient air via
temperature sensor 56 in FIG. 1 and measures the temperature of the
coolant a the temperature sensor in the coolant control system 24.
The CPU 20 then computes the power factor corresponding to the
particular segment of the PCR protocol being implemented and makes
calculations for the power factor where the power factor is the
total power needed to move the block temperature from its current
level to the temperature level specified by the user via a
setpoint.
[0062] After the required power to be applied to each of the three
zones of the heater 156 is calculated, another calculation is made
regarding the proportion of each half cycle of input power which is
to be applied to each zone in some embodiments. In one embodiment
described below, the calculation mode is how many half cycles of
the total number of half cycles which occur during a 200
millisecond sample period are to be applied to each zone. In the
alternative embodiment symbolized by FIG. 3A, the computer
calculates for each zone, the position of the dividing line 166 in
FIG. 3A. After this calculation is performed, appropriate control
signals are generated to cause the power supplies for the
multi-zone heater 156 to do the appropriate switching to cause the
calculated amount of power for each zone to be applied thereto.
[0063] In alternative embodiments, the multi-zone heater can be
implemented using a single film heater which delivers uniform power
density to the entire sample block, plus one or two additional film
heaters with only one zone apiece for the guard bands. These
additional heaters are superimposed over the single film heater
that covers the entire sample block. In such an embodiment, only
the power necessary to make up the guard band losses is delivered
to the additional heater zones.
[0064] The foregoing description illustrates how the sample block
temperature may be controlled to be uniform and to be quickly
changeable. However, in the PCR process, it is the temperature of
the sample reaction mixture and not the block temperature that is
to be programmed. In accordance with various embodiments of the
present teachings, the user specifies a sequence of target
temperatures for the sample liquid itself and specifies the
incubation times for the sample liquid at each of these target
temperatures for each stage in the PCR process. The CPU 20 then
manages the sample block temperature so as to get the sample
reaction mixtures to the specified target incubation temperatures
and to hold the sample mixtures at these target temperatures for
the specified incubation times. The user interface code run by the
CPU 20 displays, at all stages of this process, the current
calculated sample liquid temperature on the display of terminal
16.
[0065] The difficulty with displaying an actual measured sample
temperature is that to physically measure the actual temperature of
the reaction mixture requires insertion of a temperature measuring
probe therein. The thermal mass of the probe can significantly
alter the temperature of any well in which it is placed since the
sample reaction mixture in any particular well is often only 100
microliters in volume. Thus, the mere insertion of a temperature
probe into a reaction mixture can cause a temperature gradient to
exist between that reaction mixture and neighboring mixtures. Since
the extra thermal mass of the temperature sensor would cause the
reaction mixture in which it is immersed to lag behind in
temperature from the temperatures of the reaction mixtures in other
wells that have less thermal mass, errors can result in the
amplification simply by attempting to measure the temperature.
Accordingly, the instrument described herein calculates the sample
temperature from known factors such as the block temperature
history and the thermal time constant of the system and displays
this sample temperature on the display.
[0066] FIG. 5A illustrates schematically an example of operating
the sample block at ramp rates that cause the sample temperature to
either overshoot or undershoot setpoint targets set in the thermal
protocol. Typically, this occurs when the tuning parameters in the
thermal protocol have not been optimized, improperly set, or have
been applied to a different temperature range in the PCR process.
For example, the tuning parameters suitable for denaturation
incubation 170 in FIG. 3C may not incur sample temperature
overshoot at this stage, however, when applied to hybridization
incubation 172 portion of the PCR process these parameters may
incur sample temperature undershoot. Indeed, FIG. 5A illustrates
use of tuning parameters that incur both overshoot 708 and
undershoot 710 of the sample temperature though in practice one or
the other may occur independently and/or at various stages in the
PCR protocol.
[0067] In practice, it may be desirable or acceptable for the block
temperature 702 to be configured to overshoot the target setpoint
temperature as long as the sample temperature does not do the same
(or at least to a lesser degree). Rapid changes in block
temperature 702 achieved with a high target block ramp rate may be
used to urge the sample temperature to reach the target setpoint
targets in a timely manner in accordance with the particular
thermal protocol for PCR. Further, the block temperature 702 may be
configured to overshoot/undershoot the target setpoint targets in
order for the sample temperature 704 to reach target setpoint 1,
target setpoint 2 or any other setpoint in the protocol in a more
rapid manner without adverse effects on the sample amplification or
reaction. In some cases, such overshooting/undershooting may be
used to improve the thermal performance or rate of heating/cooling
of the samples which may be most efficiently accomplished by
reducing or minimizing sample overshoot/undershoot while permitting
some degree of block temperature overshoot/undershoot.
[0068] In the illustrated example, the sample temperature 704
reaches target setpoint 2 during a heating cycle at time 706 but
exceeds the specified temperature by overshoot amount 708 as the
block temperature 702 has been sustained over a time period. In
terms of the PCR process, the overshoot amount 708 may be
undesirable or not deliver the requisite accuracy for the specified
thermal protocol in relation to the sample temperature. This may
further cause a reduction in sample amplification, loss of
amplification fidelity, reduction in accuracy of the overall
experiment, or a general failure of the PCR process. In some
instances, it may also increase the overall processing time needed
to complete a full PCR cycle.
[0069] Similarly as shown by way of example the block temperature
702 may operate to cool the sample temperature at a rapid rate to
achieve target setpoint 1 in accordance with a user specified
thermal protocol. Once again, the block temperature 702 may
undershoot target setpoint 1 while transitioning the sample
temperature to a desired level for hybridization incubation 172 per
the PCR process. Instead of overshooting, the sample temperature
704 may instead undershoot the target setpoint 1 with respect to
the specified thermal protocol and incur a similar set of resulting
problems.
[0070] Aspects of the present teachings use an adaptive thermal
block temperature control to minimize, reduce or potentially
eliminate the sample temperature overshooting/undershooting of the
target setpoints. As illustrated in FIG. 5B, a block temperature
712 operates with a rapid ramp rate to achieve the sample
temperature 714 timing specified in the thermal protocol. CPU 20
uses a current block and sample temperature ramp rate to generate
an expected sample time interval (B), as depicted in FIG. 5B, that
it will take for the sample temperature to reach the target
setpoint 2. CPU 20 also determines an synchronization time interval
(A) offset from expected sample time interval (B) as determining
the adaptive window. In one implementation, the synchronization
time interval (A) may be tuned by a user based on consideration of
the PCR process and the particular sample being amplified.
[0071] In various embodiments, the adaptive window defines a time
period that the block temperature 712 is increased or decreased in
order to reach the target setpoint at the same time as the sample
temperature 714. Overshoot or undershoot is effectively reduced or
eliminated as the block temperature 712 is at substantial
equilibrium with the sample temperature 714 before entering the
next portion of the PCR process. After entering the adaptive window
in FIG. 5B, it can be seen that the block temperature may increased
or decreased relatively in small amounts or as desired to help
maintain the temperature gradient does not pull the sample
temperature 714 into either an undesired overshoot or undershoot
condition.
[0072] FIG. 6 is a flowchart diagram of the operations for
performing the adaptive thermal block control in accordance with
aspects of the present teachings. One embodiment of the present
teachings initially determines a current temperature ramp rate for
a sample being processed in a thermal block (802). The sample
temperature ramp rate may be determined empirically through various
sensors in the system or may be set according to a variety of
predetermined parameters.
[0073] Next, the current temperature ramp rate for the sample is
used to predict an expected time interval that the sample will
reach the target setpoint temperature (804). This expected time
interval is typically based upon the current temperature ramp rate,
sensor values returned by the system, parameter settings and other
relevant values obtained during operation of the thermal
cycler.
[0074] The thermal block holding the sample operates according to a
block ramp rate and the predicted time interval for the target to
reach the target setpoint temperature (806). In one implementation,
the adaptive thermal block temperature control begins when the
block temperature enters within the adaptive window previously
described. Alternatively, the adaptive thermal block temperature
control may begin sometime before the adaptive window if the block
temperature behavior and sample temperature can be more accurately
predicted and correlated together.
[0075] In one embodiment, sampling of the block temperature, sample
temperature, ambient temperature and other measurements are made
approximately 20 times per second. It will be appreciated, however,
that sampling may occur with greater or lesser frequencies as
configured or desired. In particular, aspects of the present
teachings the sampling process repeats the determination of the
current temperature ramp rate for the sample as the thermal
protocol for PCR is performed in real-time (808).
[0076] The sample temperature ramp rate results are used to provide
an improved degree of precision and control over the sample
temperature through closer control of the block temperature.
Aspects of the present teachings determine if the predicted time
interval for the sample to reach the next target setpoint
temperature has changed based on the measured temperature ramp rate
(810). This information is useful for directing that the block
temperature returns to the setpoint temperature at approximately
the same time as the sample temperature. For example, if the sample
temperature ramp rate has increased during heating then the block
temperature may drop more rapidly to ensure the sample temperature
does not substantially overshoot the setpoint. Similarly, if the
sample temperature drops more quickly than expected or desired
during a cooling region of the thermal protocol then the block
temperature may be configured to increase more rapidly to avoid the
sample temperature from reaching an undershooting condition.
[0077] Depending on change in the predicted time interval for the
sample to reach the setpoint temperature, the block ramp rate may
be increased or decreased for cooling or heating as desired.
Accordingly, the predicted time interval and the target block ramp
rate both or individually may be modified to maintain the block
temperature and the sample temperature such that both reach the
target setpoint temperature at approximately the same time (812).
Such a control process is illustrated in FIG. 5B which depicts the
result of applying the adaptive thermal block temperature control
in accordance with aspects of the present teachings. As noted
above, enhanced temperature control is achieved in both heating an
cooling steps substantially avoiding both over-shooting and
under-shooting of sample temperatures to improve PCR processes.
[0078] Having thus described various implementations and
embodiments of the present teachings, it should be noted by those
skilled in the art that the disclosures are exemplary only and that
various other alternatives, adaptations and modifications may be
made within the scope of the present teachings.
[0079] Embodiments of the present teachings can be implemented in
digital electronic circuitry, or in computer hardware, firmware,
software, or in combinations thereof. Apparatus of the present
teachings can be implemented in a computer program product tangibly
embodied in a machine-readable storage device for execution by a
programmable processor; and method steps of the present teachings
can be performed by a programmable processor executing a program of
instructions to perform functions of the present teachings by
operating on input data and generating output. The present
teachings can be implemented advantageously in one or more computer
programs that are executable on a programmable system including at
least one programmable processor coupled to receive data and
instructions from, and to transmit data and instructions to, a data
storage system, at least one input device, and at least one output
device. Each computer program can be implemented in a high-level
procedural or object-oriented programming language, or in assembly
or machine language if desired; and in any case, the language can
be a compiled or interpreted language. Suitable processors include,
by way of example, both general and special purpose
microprocessors. Generally, a processor will receive instructions
and data from a read-only memory and/or a random access memory.
Generally, a computer will include one or more mass storage devices
for storing data files; such devices include magnetic disks, such
as internal hard disks and removable disks; magneto-optical disks;
and optical disks. Storage devices suitable for tangibly embodying
computer program instructions and data include all forms of
non-volatile memory, including by way of example semiconductor
memory devices, such as EPROM, EEPROM, and flash memory devices;
magnetic disks such as internal hard disks and removable disks;
magneto-optical disks; and CD-ROM disks. Any of the foregoing can
be supplemented by, or incorporated in, ASICs.
[0080] Thus, the present teachings is not limited to the specific
embodiments described and illustrated above. Instead, the present
teachings is construed according to the claims that follow and the
full scope of their equivalents thereof.
* * * * *