IntroductionTemperature cycling is the basis for all PCR, and how your thermal cycler performs is of critical importance to you and your research. Temperature, ramp rate, and hold time are the critical parameters controlled by a thermal cycler for a successful PCR reaction. A high-performing thermal cycler must have the ability to precisely control the liquid (reaction) temperature and hold time, regardless of the reaction volume. The temperature and hold-time control of a thermal cycler is even more critical during PCR optimization experiments. The thermal cycler must provide the same performance during normal PCR and during PCR optimization runs. This paper analyzes the construction of instruments that use Applied Biosystems™ VeriFlex™ Blocks temperature control technology or conventional gradient blocks and compares their performance.

What is gradient temperature control?Gradient temperature control is one of the features that helps the user conduct PCR optimization experiments to determine the optimum temperature and hold time of a PCR protocol, and do it with the minimal number of experiments. Ideally, a true gradient will exhibit a linear temperature slope across a homogenous metal block, as shown in Figure 1.

Gradient thermal cyclers on the market today are constructed with two separate heating/cooling elements below a homogenous metal block, as shown in Figure 2. This design results in the following limitations:

• On a gradient thermal cycler, the user can only set two temperatures and cannot control other temperatures across the block.

• A true linear gradient is not achievable because of heat interactions between the high and low temperature set at each end of the homogenous metal block. Instead of a linear gradient, the temperature across a homogenous metal block follows more of a sigmoidal curve, as shown in Figure 3.

VeriFlex temperature control technology for thermal cycling

APPLICATION NOTEVeriFlex temperature control technology

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Linear gradient temperature across metal block

Figure 1. Linear gradient temperature.

Figure 2. Gradient thermal cycler construction.

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65.6

70.3 73.5

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Typical gradient temperature across metal block

Figure 3. Nonlinear gradient temperature.

What is VeriFlex Blocks temperature control?Thermal cyclers that use VeriFlex technology are constructed with 3 or more separate heating/cooling elements below each of the 3 or more segmented metal blocks, as shown in Figure 4. Each pair of heating/cooling elements and segmented metal blocks are completely insulated from each other to help prevent heat interactions. As a consequence, VeriFlex technology allows for a true linear temperature slope across metal blocks, as shown in Figure 1, with the ability to set up to 6 different temperatures. The user can set each temperature zone uniquely, allowing for better control of temperature optimization.

Materials and methodsThe instruments in Table 1 were all tested using the same equipment and methods, which are described in this section.

Table 1. Instruments tested.

Model name Cat. No.

Eppendorf Mastercycler™ Nexus Gradient 6331 000.017

Eppendorf Mastercycler™ Nexus GX2 6336 000.015

Bio-Rad C1000 Touch™ 96-well Fast 185-1196

Bio-Rad T100™ 186-1096

Applied Biosystems™ ProFlex™ 1 x 96 well 4484075

Applied Biosystems™ SimpliAmp™ A24811

SensoQuest LabCycler 011-101, 012-103

Bioer GeneMax BYQ6067

Takara Dice™ Touch TP 350

Block temperature was measured using a NIST-traceable Applied Biosystems™ VeriFlex™ 96-well Temperature Verification Kit (Cat. No. 4377669) with 3 temperature probes. Each probe measures temperature at 8 positions, providing a total of 24 temperature measurements at various points on the block. An example of the temperature probe layout is diagrammed in Figure 5.

Temperatures are the average probe temperatures of each row, column, or zone.

Figure 4. VeriFlex technology construction.

Figure 5. VeriFlex Temperature Verification Kit temperature probe layout.

Table 2. Example accuracy at various set points over a 20°C range.

VeriFlex technology versus conventional gradient block accuracyEach instrument in this study was tested using its gradient or VeriFlex Blocks technology to vary temperatures. 60°C (a common annealing temperature) was used as the approximate center point, and then each block was tested with the maximum temperature range allowed by the instrument. An example of several temperature settings, actual measurements, and calculated difference is shown in Table 2.

Temperature set point1 (°C) 50.5 51.7 53.6 56.2 58.8 61.2 63.8 66.4 68.3 69.5

Block temp measurement2 (°C) 50.6 51.8 53.7 56.1 58.7 61.2 63.8 66.3 68.4 69.4

Delta3 (°C) 0.1 0.1 0.1 –0.1 –0.1 0.0 0.0 –0.1 0.1 –0.1

1. As displayed on the interface of the thermal cycler.2. As measured using the VeriFlex 96-well Temperature Verification Kit.3. See Table 3 for the largest discrepency observed across the whole temperature range

for each instrument tested.

VeriFlex technology compared to conventional gradients

ResultsMeasured temperature profi les across the metal blocks of each thermal cycler are shown in Figure 6. VeriFlex Blocks temperature profi les show true linear temperature control across the blocks. All gradient blocks show a similar nonlinear temperature profi le (red line) that gives inconsistent lane-to-lane temperature differences (blue bars), except the Bioer GeneMax thermal cycler. The inconsistent temperature differences from lane-to-lane make it challenging to determine the true optimized annealing temperature, especially when optimizing diffi cult assays.

The published values for temperature range and the number of different temperatures available for the instruments in this study, along with the maximum discrepancy between set points and the measured temperatures for each instrument are shown in Table 3.

Figure 6. Measured temperature profi les across thermal cycler metal blocks.

Table 3. Temperature accuracy of conventional gradients compared to VeriFlex technology.

Instrument Temperature rangeNumber of different

temperatures in range

Accuracy(maximum discrepancy,

set point vs. actual)Bio-Rad C1000 Touch 24°C 8 –1.2°C @ 75°CBio-Rad T100 25°C 8 +0.6°C @ 50°CSensoQuest LabCycler 40°C 12 –0.9°C @ 66.9°CEppendorf Mastercycler Nexus Gradient 20°C 12 –0.2°C @ 58.8°CEppendorf Mastercycler Nexus GX2 12°C 8 –0.4°C @ 61.8°CBioer GeneMax 30°C 6 +0.3°C @ 69°CApplied Biosystems Profl ex 25°C 6 –0.2°C @ 75°C Applied Biosystems SimpliAmp 20°C 3 0.0 @ 60°CTakara Dice Touch 24°C 12 –0.8°C @ 58.9°C

0.50

1.20

1.90

2.60 2.60 2.40

2.60 2.60

1.90

1.20

0.50

0.00

0.50

1.00

1.50

2.00

2.50

3.00

45

50

55

60

65

70

75

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-to-

lane

tem

pera

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diff

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C)

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Eppendorf Nexus 96-well gradient profile

Lane-to-lane temperature difference Gradient temperature

2.10

3.00

3.80 4.20

4.60 4.60 4.60 4.20

3.80

3.00

2.10

0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00

35 40 45 50 55 60 65 70 75 80 85

1 2 3 4 5 6 7 8 9 10 11 12 Lane

-to-

lane

tem

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diff

eren

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C)

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pera

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Lane

SensorQuest LabCycler gradient profile

Lane-to-lane temperature difference Gradient temperature

5.00 5.00 5.00 5.00 5.00

0.00

1.00

2.00

3.00

4.00

5.00

6.00

45

50

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60

65

70

75

80

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-to-

lane

tem

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C)

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Applied Biosystems ProFlex temperature profile

Lane-to-lane temperature difference Temperature

0.60

1.50

2.40

3.20

2.10

1.50

0.70

0.00

0.50

1.00

1.50

2.00

2.50

3.00

3.50

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Eppendorf GX2 64-well gradient profile

Lane-to-lane temperature difference Gradient temperature

0.75

1.41

2.40

3.36

2.64

3.12

2.64

3.60

2.40

1.44

0.24

0.00

0.50

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Takara Dice Touch gradient profile

Lane-to-lane temperature difference Gradient temperature

6.00 6.00 6.00 6.00 6.00

0.00

1.00

2.00

3.00

4.00

5.00

6.00

7.00

40

45

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70

75

80

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-to-

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Bioer GeneMax gradient profile

Lane-to-lane temperature difference Gradient temperature

10.00 10.00

0.00

2.00

4.00

6.00

8.00

10.00

12.00

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Applied Biosystems SimpliAmp temperature profile

Lane-to-lane temperature difference Temperature

1.80

3.00

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5.50 5.10

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Bio-Rad T100 gradient profile

Lane-to-lane temperature difference Gradient temperature

1.50

3.00

4.60

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4.70

3.20

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Bio-Rad C1000 Touch gradient profile

Lane-to-lane temperature difference Gradient temperature

Find out more at lifetechnologies.com/thermalcyclers

SummaryConventional gradients VeriFlex technology

One homogenous metal block Multiple segmented metal blocks

Only two temperature set points, at both ends of single metal block

Settable temperatures for each segmented metal block

Temperature control at both ends of metal block Independent and precise temperature control of each segmented metal block

Temperatures across metal block are nonlinear Temperature across multiple segmented metal blocks can be linear

Temperature hold time varies across metal block during gradient experiments

Designed to set temperature hold times accurately

Difficult to resolve small temperature differences during gradient experiment

Enables independent and precise temperature control of each segmented metal block easily permits resolution of temperature differences of 0.1°C

Temperature accuracy in gradient mode may not match the temperature accuracy of normal mode

Temperature accuracy maintained whether in VeriFlex mode or normal mode

Additional VeriFlex technology applicationsVeriFlex Blocks provide a better-than-gradient approach to PCR optimization. VeriFlex Blocks come in multiple zonal Peltier blocks that are controlled individually by a temperature controller. Each Peltier block is physically isolated using heat insulator material to minimize temperature interference among adjacent Peltier blocks. The result enables precise control over multiple temperature zones.

• Offers highly accurate determination of optimal annealing temperature to help eliminate guesswork

• Helps save time and offers the ability to run multiple reactions with different annealing temperatures in a single PCR experiment run

• Use your thermal cycler as an incubator with multiple precise incubation temperatures

For Research Use Only. Not for use in diagnostic procedures. © 2015 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified. Bio-Rad, C1000 Touch, and T100 are trademarks of Bio-Rad Laboratories, Inc. Eppendorf and Mastercycler are trademarks of Eppendorf AG. Takara and Dice are trademarks of Takara Holdings, Inc. CO29782 0415