As we all know, traditional gradient PCR can only optimize one temperature at a time, and you have to manually adjust each step—annealing, denaturation, extension... one by one.
But don’t worry—Apices’s PCR system is here to solve this tricky problem for you!
It allows you to optimize temperatures for two steps in a single run, completing the optimization program in just one go.
So, what exactly is this powerful feature that makes it all possible?
Let’s find out now!
To begin, we need to understand the meaning and application of linear gradient temperature and two-dimensional (2D) gradient temperature.
1. What is Linear Gradient Temperature?
In PCR reactions, the annealing temperature is a key factor that determines success.
Linear gradient refers to a temperature distribution that increases or decreases linearly across each row of wells in the PCR block. In simpler terms, it means starting from a fixed temperature and increasing or decreasing it step by step along a straight line.
This allows you to quickly identify optimal reaction conditions in a single run.
Because different DNA fragments have different optimal annealing temperatures, setting up a range of linear gradient temperatures enables you to determine the best condition for efficient amplification—specifically, the one that yields the highest expression—all in a single PCR run.
2. What is 2D Gradient Temperature?
2D gradient PCR is an advanced and improved PCR technique that utilizes two temperature gradients to optimize PCR reactions. This approach enhances both the specificity and purity of PCR products.
It is based on how the binding efficiency between primers and templates varies under different temperature conditions. By gradually increasing or decreasing factors such as primer concentration and temperature, the system optimizes thermal gradients to enable specific amplification under the most favorable temperature settings.
This method helps prevent non-specific hybridization or products caused by insufficient primer binding. By fine-tuning temperature-related parameters, it improves the specificity and purity of the amplified products.
Compared to traditional PCR, 2D gradient PCR creates a matrix of different reaction temperatures across both space and time, forming 96 unique temperature combinations. This allows researchers to explore and identify the most optimal temperature conditions for amplification.
Applications of Linear Gradient
Linear gradient temperature is primarily used for optimizing the annealing temperature of unknown DNA sequences. It is suitable for a wide range of fields, including:
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Forensic science
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Medical diagnostics
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Food industry
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Molecular biology
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Biotechnology
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Environmental science
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Microbiology
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Clinical diagnostics
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Epidemiology
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Genetics
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Gene chips
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Genetic testing
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Gene cloning
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Gene expression studies
Even without setting a gradient, the system can function as a standard PCR instrument, offering flexibility in usage.
Applications of 2D Gradient
Traditional gradient PCR instruments typically only allow variability in annealing temperature, while denaturation temperature is fixed at 95°C and extension temperature at 72°C by default. However, amplification efficiency can also be significantly affected by variations in denaturation temperature.
For example, a specific DNA fragment may yield the brightest band at 93.0°C, while another might perform best at 93.6°C. In such cases, optimizing denaturation temperature is essential.
This is where 2D gradient PCR comes in—it allows simultaneous optimization of two temperature parameters (e.g., annealing and denaturation) in a single run, streamlining the entire optimization process.
01 Linear Gradient
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In the annealing temperature step, click on Advanced Settings and switch the gradient mode to “Linear”.
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Set the base temperature and the gradient increment.
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Define the temperature increment and its starting point.
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Set the time increment and its starting point.
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Configure the temperature ramp rate (rate of temperature change).
02 2D Gradient
In practical applications, researchers typically predefine the range of denaturation and annealing temperatures based on factors such as the size of the gene fragment, the Tm values of the primers, and specific experimental requirements.
During the experiment, the 2D gradient PCR instrument automatically configures and adjusts the gradient temperature settings, creating the most suitable thermal conditions for efficient and specific PCR amplification.
Two-Dimensional Gradient Step Setting on the RePure-C Intelligent 2D Gradient PCR Instrument:
（1）Add the desired reaction steps to the temperature control program.
（2）Set the gradient temperature ranges:
- Vertical (Y-axis) gradient range: 0.1–30°C
- Horizontal (X-axis) gradient range: 0.1–42°C
（3）Configure additional parameters based on experimental requirements, including:
- Temperature increment and its starting point
- Time increment and its starting point
- Temperature ramp rate (rate of temperature change)
01 Linear Gradient
（1）Quickly Identifies the Optimal Annealing Temperature
Linear gradient PCR allows simultaneous amplification at multiple annealing temperatures, helping to rapidly determine the optimal condition.
（2）Shortens Experiment Time
With just one experiment, the ideal annealing temperature for a specific reaction system can be identified, significantly improving the efficiency of PCR research.
（3）Improves Efficiency and Reduces Costs
By reducing experiment duration and sample usage, linear gradient PCR increases experimental throughput and lowers overall costs.
02 2D Gradient
（1）Improves Specificity of PCR Reactions
Changes in primer-template binding efficiency under different 2D gradient temperatures enhance specificity and reduce non-specific hybridization products.
（2）Increases Product Purity
By optimizing the reaction for each molecular target, the purity of amplified DNA products is significantly improved.
（3）Detects Gene Point Mutations
Specific primers designed for known mutation sites can be used to amplify target gene fragments. These products can then be sequenced to detect mutations.
（4）Analyzes Genetic Diversity Between Populations
Using 2D gradient PCR followed by sequencing allows for the identification of mutation sites and comparison of genetic diversity across different populations.
（5）Simple Operation
Only a single reaction system setup is required to perform a full 2D gradient optimization.
（6）Enhances Accuracy of Positive Detection
2D gradient PCR provides up to 96 unique temperature combinations in a single run, greatly reducing the chance of cross-contamination and false positives, thus improving the accuracy of true positive detection.
（7）Reduces Experiment Time
Since the optimal reaction temperatures (both annealing and denaturation) can be determined in a single run, it eliminates the need for multiple rounds of temperature optimization.
Summary
Compared to linear gradients, 2D gradient PCR not only enables exploration of 96 temperature combinations in a single run, but also achieves faster and more accurate identification of optimal annealing and denaturation temperatures. This ensures that amplification occurs under the most favorable thermal conditions, enhancing primer-template specificity and amplification efficiency.
Furthermore, it enables detection of point mutations and genetic diversity analysis, offering valuable tools for the prevention, diagnosis, and treatment of certain diseases in the future.
All RP-series gradient PCR instruments from Meide Technology are equipped with linear gradient functionality.
As for 2D gradient capability, several models already support it, including: RePure-C, RePure-D, and RePure-D(P).