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Thermal Cycling Optimization
by K Chico, Jessica Piczon
A polymerase chain reaction (PCR) thermal cycler is an instrument that amplifies target DNA sequences. Thermal cyclers can effectively regulate temperatures in a cyclical program. To understand thermal cycling optimization, one must first understand the thermal cycler stepwise process.
In optimizing thermal cycling conditions, it is necessary to understand the impact of temperature and duration on each step. For denaturing, incomplete denaturation of the double stranded (ds) DNA could allow the DNA strands to snapback, reducing the product yield. If denaturation is performed at too high of a temperature for too long, enzyme activity will be lost in latter steps.
In annealing, the temperature and step duration are dependent upon base composition, as well as length and concentration of the primers and template. Optimizing the annealing temperature could enhance the PCR and should be considered in combination with additives, if necessary. A higher annealing temperature may increase the discrimination against incorrectly annealed primers, ultimately reducing mis-extension of non-targets. Stringent annealing temperatures, especially during the first several cycles of the annealing step, can also help increase specificity.
Importantly, Taq DNA polymerase is active at the annealing temperature, so primer extension can occur during the annealing step. In the extension step, the efficiency of primer extension will depend on a number of factors. At a target temperature of 72 °C, extension can vary from 35 -100 nucleotides per second depending on the buffer, pH, salt concentration, and the nature of the DNA template. Lastly, the optimum number of cycles to be performed in a PCR depends mainly on the starting concentration of the target DNA, if other parameters are already optimized. An excessive number of cycles can increase the amount and complexity of nontarget products, while too few cycles can result in low product yield.
Various spinoff methods of PCR have been developed to help optimize thermal cycling. In hot start PCR, the initial denaturation time increases dramatically and is often used alongside additives to aid in temperamental amplicon formation. Comparatively in touchdown PCR, the annealing step starts at 10°C the Tm of the primers and is successively lowered every second cycle (1-2°C) for 20-25 cycles. Then, annealing is carried out for 20-25 cycles at an annealing temperature 5 °C below the Tm. Similarly, stepdown PCR and slowdown PCR are both modifications of touchdown PCR, where the temperatures, durations, and the number of cycles is adjusted.
Polymerase Chain Reaction: Basic Protocol Plus Troubleshooting and Optimization Strategies
Thermal Cycling Parameters & Optimization
Original created on November 22, 2023, last updated on November 22, 2023
Tagged under: Thermal cycling, PCR, amplification
Thermal cycling steps, including common settings and temperature ranges. Figure made in BioRender.
A polymerase chain reaction (PCR) thermal cycler is an instrument that amplifies target DNA sequences. Thermal cyclers can effectively regulate temperatures in a cyclical program. To understand thermal cycling optimization, one must first understand the thermal cycler stepwise process.
In optimizing thermal cycling conditions, it is necessary to understand the impact of temperature and duration on each step. For denaturing, incomplete denaturation of the double stranded (ds) DNA could allow the DNA strands to snapback, reducing the product yield. If denaturation is performed at too high of a temperature for too long, enzyme activity will be lost in latter steps.
In annealing, the temperature and step duration are dependent upon base composition, as well as length and concentration of the primers and template. Optimizing the annealing temperature could enhance the PCR and should be considered in combination with additives, if necessary. A higher annealing temperature may increase the discrimination against incorrectly annealed primers, ultimately reducing mis-extension of non-targets. Stringent annealing temperatures, especially during the first several cycles of the annealing step, can also help increase specificity.
| Assaywise Letters: |
Importantly, Taq DNA polymerase is active at the annealing temperature, so primer extension can occur during the annealing step. In the extension step, the efficiency of primer extension will depend on a number of factors. At a target temperature of 72 °C, extension can vary from 35 -100 nucleotides per second depending on the buffer, pH, salt concentration, and the nature of the DNA template. Lastly, the optimum number of cycles to be performed in a PCR depends mainly on the starting concentration of the target DNA, if other parameters are already optimized. An excessive number of cycles can increase the amount and complexity of nontarget products, while too few cycles can result in low product yield.
Table 1. Overview of the basic steps in the qPCR cycling reaction
| Step ▲ ▼ | Temperature ▲ ▼ | Time ▲ ▼ | Process ▲ ▼ |
| Denaturation | 95°C | ∼20 to 30 seconds | Double-stranded DNA (dsDNA) template is heated to high temperature. This disrupts the hydrogen bonds between the complementary base pairs causing dsDNA to separate into single-stranded DNA (ssDNA).
|
| Primer Annealing | 48 to 72°C | ∼20 to 40 seconds | After denaturation, the reaction temperature is lowered to ∼48 to 72°C. This promotes the binding of forward and reverse primers to each of the ssDNA templates and the subsequent binding of DNA polymerases to the primer-template hybrid.
|
| Extension | 68 to 72°C | ∼1 to 2 minutes | After annealing, the reaction temperature is raised to ∼68 to 72°C. This enables DNA polymerase to extend the primers, synthesizing new DNA strands complementary to the ssDNA template in the 5’ to 3’ direction. |
Various spinoff methods of PCR have been developed to help optimize thermal cycling. In hot start PCR, the initial denaturation time increases dramatically and is often used alongside additives to aid in temperamental amplicon formation. Comparatively in touchdown PCR, the annealing step starts at 10°C the Tm of the primers and is successively lowered every second cycle (1-2°C) for 20-25 cycles. Then, annealing is carried out for 20-25 cycles at an annealing temperature 5 °C below the Tm. Similarly, stepdown PCR and slowdown PCR are both modifications of touchdown PCR, where the temperatures, durations, and the number of cycles is adjusted.
Products
Table 2. Possible ROX Reference and reporter dye combinations for multiplex qPCR assays.
| Instrument ▲ ▼ | Reference Dye ▲ ▼ | Reporter Dye 1 ▲ ▼ | Reporter Dye 2 ▲ ▼ | Reporter Dye 3 ▲ ▼ | Reporter Dye 4 ▲ ▼ |
| ABI PRISM® 7700 | ROX | 6-FAM | 6-TET | - | - |
| ABI PRISM® 7000 and 7900 Applied Biosystems® 7300 StepOnePlus™ | ROX | 6-FAM | 6-TET | 6-HEX | - |
| Applied Biosystems® 7500 | ROX | 6-FAM | 6-TET | 6-HEX | Tide Fluor™ 3 iFluor® 647 Alexa Fluor 647 Cy5 |
References
Polymerase Chain Reaction: Basic Protocol Plus Troubleshooting and Optimization Strategies
Thermal Cycling Parameters & Optimization
Original created on November 22, 2023, last updated on November 22, 2023
Tagged under: Thermal cycling, PCR, amplification