Studying DNA is like unraveling the secrets hidden in a mystery book. Each letter, each sequence, forms the basic building blocks of life. However, the methods used to open the pages of this book determine the accuracy of the information you will obtain. Two methods are used. Dye-based methods offer an approach that stains the entire double-stranded DNA and creates an overall glow. Like a flashlight shining on a crowded party, this method illuminates the entire DNA sequence. But here is the problem: Finding the target DNA is like looking for a specific person in a crowd. Because everything is illuminated, it can be difficult to know which sequence is important. This is where the probe-based methods commonly used in qPCR (quantitative PCR) and dPCR (digital PCR), the super tools of molecular biology, were developed to solve this problem. These state-of-the-art probes are like intelligent guides that know exactly where to bind in a DNA sequence. They lock on to a specific target and mark only that sequence, producing a much sharper and more sensitive signal. So probe-based methods allow you to find the answer directly in the complex sea of DNA letters. In other words, while dye-based methods give a general idea, probe-based methods offer the guarantee of getting to the right address. Let’s take a closer look at how these probes work and why they are revolutionizing the world of science.
Probes The DNA you are looking for has been found!
It is not easy to specifically recognize and analyze a DNA sequence. That is why special pieces of DNA called probesare used. When the probes find the target sequence, they fit over it like a “lock”. But this process is more complex than solving an ordinary jigsaw puzzle. The probes also work like “detectives”, marking a specific target in the DNA sequence and showing scientists where it is. Different types of probe can be chosen according to your expectations in the study. Let’s get to know the different probe types briefly.
Dual-labeled Probes (TaqMan Probes)
Dual-labeled probes are like a balance where light and darkness meet. These probes have two special molecules at their ends: a fluorescent reporter and aquencher. The fluorescent reporter is a molecule with the potential to emit light; like a flashlight, it “lights up” whether DNA is bound or not. The quencher absorbs the light emitted by the reporter and prevents it from escaping, like a curtain in front of a flashlight. So, under normal conditions, no light can be seen. But during PCR, the magic begins. The probe binds to its target in the DNA and is cleaved by the enzyme polymerase. This cleavage separates the reporter from the quencher and the reporter is freed. At that moment, the reporter molecule begins to emit fluorescence, just as the light from a lantern shines out when the curtain is opened. This glow “lights up”, letting scientists know that the target DNA has been found and can be analyzed. Thanks to these properties, double-labeled probes are reliably used in many fields, from diagnosing genetic diseases to detecting viruses. For example, COVID-19 tests use such probes to detect the RNA of the virus.
Double-quencher (Double With extinguisher) Probes Clearer and Sharper Results
Dual-quencher probes are like a special lens that a detective uses to get a sharper and clearer view. They differ from ordinary dual-labeled probes in that they contain two quencher molecules. Normally, a reporter (the molecule that produces fluorescent light) and a quencher (the molecule that absorbs this light) are side by side. The quencher is so close to the reporter that the light from the reporter is absorbed before it is emitted and cannot reach the outside. So, even if the reporter is present, no signal is seen. With dual-quencher probes, however, a second quencher is added to the system, further shortening the distance between the reporter and the quencher. What does this achieve? The shorter the distance, the more unwanted small light that can escape from the reporter is completely absorbed. Thus, unnecessary background light (fluorescence) is largely eliminated. As a result, the signal becomes clearer, stronger and more reliable. This is a great advantage, especially when analyzing long DNA sequences, because the sharpness of the signal makes it easier to identify the right target. Double-quenched probes are like a focusing tool that eliminates blurring when detecting DNA. They are particularly preferred in applications such as the detection of genetic variations (SNPs) and the analysis of rare mutations.
MGB Probes: Short, Sharp and Strong
Molecules called Minor Groove Binders (MGB) grip the DNA by holding tightly to the small grooves on its surface. MGB grips the tiny folds of the DNA like a key fitting snugly into a lock, allowing the probe to bind to the DNA much more strongly. This tight grip increases the melting temperature (Tm) of the probe, meaning it is harder for the probe to detach from the DNA sequence.
What does this mean? Normally, for a probe to bind to DNA, the sequences need to be long so that the binding is strong. But MGB probes bind so strongly to DNA that the sequence doesn’t need to be long. Even short sequences become very stable and perfectly recognize the target sequence. In short, thanks to MGB probes, scientists can confidently detect the target DNA sequence with shorter and more precise probes.
This is a great advantage, especially in complex systems such as multiplex PCR. Multiplex PCR is a process where multiple DNA sequences are analyzed simultaneously and accuracy is vital. MGB probes provide accurate and reliable results even when analyzing complex sequences because they clearly recognize the target sequence. Therefore, MGB probes are frequently preferred especially for amplification of regions with high GC content.
LNA Probes: To Solve the Toughest Mysteries in DNA
Some DNA sequences are more complex than others and detecting small changes in these sequences is not always easy. In particular, genetic variations– when even a single letter in DNA is different (these changes are called polymorphisms) – are a challenge, like finding the missing piece of a puzzle. For such challenges, scientists use special probes called Locked Nucleic Acid(LNA). The power of LNA probes comes from the fact that they are different and more robust than ordinary DNA building blocks. While normal DNA forms flexible and loose bonds, LNA building blocks form tight and stable bonds, like a locked door. This structure allows LNA probes to attach much more strongly to the DNA sequence. Just like a strong magnet sticks firmly to a metal surface, LNA probes grip the target DNA tightly. What does this achieve? LNA probes can detect even very small changes in the DNA sequence. For example, if only a single base (one of the letters A, T, C, G) is different in a sequence, the LNA probe will pick up this difference and signal correctly. This makes LNA probes ideal for highly sensitive and accurate applications, such as the detection of genetic diseases. LNA probes work like a detective, capturing even the finest details in DNA, allowing us to solve difficult targets with confidence. They are frequently used in applications such as multiplex PCR, detection of single base polymorphisms (SNPs), microRNA analysis and gene expression studies. LNA probes also have the potential to be used in antisense technology studies.
Molecular Beacons: The Power of Hairpin Structure
Molecular beacons get their name from their unique structure; they curl like a hairpin. These special probes are like a sensitive detector that tracks DNA and only activates when it finds its target. Normally, molecular beacons form a closed structure in the shape of a hairpin. At the ends of this closed structure are a fluorescent reporter and a quencher. The reporter molecule wants to emit light, but the quencher is so close to it that it absorbs it immediately. Therefore, when the beacon is off, fluorescence cannot be seen through the quencher. When the beacon is fully aligned to the target, the hairpin structure opens and the reporter and quencher move away from each other. At that moment, the reporter molecule is released and starts to emit fluorescent light. This signal indicates that the target DNA has been successfully detected. The most important feature of molecular beacons is that they are extremely sensitive. They can even detect a change in a single DNA base, such as the letter G instead of A. This is a huge advantage, especially in genetic analysis, where even very small differences are important. Beacons wait until they find the right target, and when they do, they signal just like a light bulb going on. They are widely used in areas such as gene expression analysis, pathogen detection and genetic diagnosis.
Tm(Melting Temperature) Enhancing Modifications: DNA Specific Tuning
The secret to success in PCR is to get the DNA to open at the right temperature. Tm (melting temperature) is the temperature at which the double helix structure of DNA dissolves and becomes a single helix. In other words, it is the temperature at which the two DNA strands are separated. The Tm value affects how tightly the primer binds to the target DNA. If the Tm value of the primer is too low, the primer will not bind strongly enough to the target DNA, which can lead to false results. Therefore, depending on the needs of your study, it is sometimes necessary to increase the Tm value to increase the binding strength of the primer to the target DNA. This is where “Tm-enhancing modifications” can be a great help. These modifications aim to increase the Tm value by adding special chemical groups to the structure of the primer. For example, modifications such as propynyl-dC(pdC) and propynyl-dU(pdU) strengthen the bonds between DNA and primer. These modifications increase hydrogen bonds between DNA molecules, allowing the double helix structure to unwind at higher temperatures. Just as a magnet sticks more firmly to a stronger magnet, modified primers bind more firmly to the target DNA. pdC increases the Tm value by about 2.8°C, while pdU increases it by 1.7°C.
As for the benefits of Tm-enhancing modifications;
– It enables primers to bind more specifically and strongly to the target DNA, reducing the risk of false positive or false negative results.
– Increases the sensitivity of PCR by enabling detection of even low amounts of target DNA.
– It gives better results in challenging PCR applications, for example when amplifying regions with high GC content or when multiple targets are amplified simultaneously, such as multiplex PCR.
Conclusion: Decoding DNA
Molecular biology is the process of decoding DNA, and probes are scientists’ most reliable assistant in this process. With technologies such as qPCR and dPCR, these probes reveal genetic information in the most accurate and precise way. Advanced probe types, especially innovations such as LNA and MGB, ensure success even with complex and difficult targets. Therefore, analyzing DNA has become more precise, more reliable and faster.
In conclusion, the probes used in qPCR and dPCR technologies are the most powerful tools at our disposal to unlock the secrets of DNA. Each probe type, with its unique advantages, opens new doors for researchers, clinicians and the entire scientific world. These molecular keys are revolutionizing science by enabling us to understand the codes of life.
Probe By : Letgen Biotechnology & Microsynth
In our mission to provide the best service to the scientific community, Letgen Biotechnology has a strong collaboration with Microsynth, one of Europe’s leading nucleic acid synthesis and analysis companies. Thanks to this partnership, we are able to offer researchers the highest quality probes at competitive prices.
Why? Microsynth Should You Choose Probes?
Microsynth probes have a number of advantages to ensure you get the most accurate and reliable results for your genetic research:
Presented in collaboration with Letgen Biotechnology and Microsynth, these high quality probes will accelerate your scientific discoveries and open new horizons in your research. With our competitive prices, you can get the best quality without breaking your budget. Letgen Biotechnology and Microsynth are always with you on your path to scientific success.
