Fluorescence qPCR refers to the method of adding fluorescent chemicals to the PCR reaction system and using the accumulation of fluorescent signals to monitor PCR amplification products in real-time and perform quantitative analysis. According to the different fluorescent chemicals used, qPCR can be divided into the fluorescent dye method (i.e., SYBR Green I dye) and the fluorescence-quenching probe method (i.e., TaqMan probe).
Fluorescence-quenching probes have gradually become the mainstream method of qPCR due to their high sensitivity and specificity, and the decrease in the synthetic price of fluorescence-quenching probes, so that the cost of single detection is very low.
To enhance the detection capability of fluorescent qPCR and its applicable scope, several probe modifications are used to enhance detection capability of fluorescent qPCR and its application scope. The following article will introduce the types of probe modifications in real-time PCR detection.
TaqMan assays have long been widely used as the gold standard for real-time PCR. Unlike dye-based real-time PCR, TaqMan assays add specific fluorescent-quenching probes to improve the specificity of fluorescent detection.
Before PCR begins, the TaqMan probe is intact and can bind to the specific complementary pair of the template sequence. When the probe is intact, the reporter group and the quencher are within a certain distance and spectrally matched, which can promote the occurrence of FRET, resulting in the quenching of the fluorescent signal of the reporter group.
TaqMan probe method fluorescent quantitative PCR, real-time fluorescent detection is performed by introducing a TaqMan probe into a conventional PCR system. This TaqMan probe is an oligonucleotide that binds to the amplification region of the PCR primer, and as the PCR reaction proceeds, DNA polymerase extends to the probe and hydrolyzes it to generate a signal. Therefore, TaqMan probes are one of the important members of hydrolysis probes.
In addition to the fluorophore and quencher group at both ends of the MGB probe, the 3′ or 5′ end of the probe is also connected with a Minor groove binding (MGB), wherein the labelled quencher group is a non-fluorescent quencher (NFQ), and MGB is mostly a small molecule tripeptide, which can non-covalently bind to the minor groove of double-stranded DNA to stabilize the DNA structure.
MGB probes include three types of MGB-TaqMan probes, MGB-Pleiades probes, and MGB-Eclipse probes. The TaqMan-MGB probe is based on the TaqMan probe, and MGB is attached to its 3′ end, to act as a hydrolysis probe, it is hydrolyzed by the Taq enzyme during the amplification process to release a fluorescent group to generate a signal. The 5′ end of the Pleiades-MGB probe is labelled with a fluorophore and connected with MGB, and the 3′ end is labelled with a quencher group; the Eclipse-MGB probe is labelled with a quencher group at its 5′ end and connected with MGB, 3′ End-labeled fluorophore.
Both the Pleiades-MGB probe and the Eclipse-MGB probe are hybridization probes (different from hydrolysis probes), and the MGB attached to the 5′ end can prevent it from being hydrolyzed by the Taq enzyme, so the two probes are hybridized with the target sequence., the fluorophore and the quencher group are relatively far apart, resulting in a fluorescent signal (the luminescence mechanism is like that of a molecular beacon).
Double Quenching Probe
As the name suggests, the “double quenching” probe has two “fire lines” to ensure the quenching of the fluorophore: based on the common 5′ nuclease hydrolysis probe, in addition to the common quenching group at the 3′ end of the probe group (for example, Black Hole Quencher, etc.), and an additional quencher group is added in the middle of the probe.
Take the IDT double quenching probe as an example, which attaches a second quenching group to the 9th or 10th base of the probe: ZEN™️ quencher or TAO™️ quencher.
ZEN dual quencher probes contain a 5′ fluorophore, a 3′ Iowa Black™ FQ (IBFQ) quencher, and a proprietary built-in ZEN quencher, the most used 5′ fluorophore Groups include FAM, TET, Yakima Yellow, SUN, or HEX.
TAO dual quencher probes carry a Cy 5 fluorophore at the 5′ end, an Iowa Black RQ (IBRQ) quencher at the 3′ end, and a proprietary built-in TAO quencher.
The double quenching probe has double guarantees. In addition to the common quenching group at the 3′ end of the probe, second quenching group is added inside, which can reduce the background signal and improve the signal-to-noise ratio.
The length of the traditional probe is generally 20~30bp, and the double quenching probe can be designed to be longer, reaching 40 bases.
Double quenching probes can reduce the crosstalk of fluorescent signals in multi-channel detection in multiplex qPCR detection.
The double quenching probe can also enhance the stability of the double-stranded structure, prevent the enzymatic cleavage of exonuclease, and has no cytotoxicity, making it suitable for in situ hybridizations of anti-miRNA sequences and miRNA, mRNA, etc.
Locked Nucleic Acid Probe
Locked nucleic acid (LNA) is a class of modified high-affinity RNA analogues whose 2′-O and 4′-C positions are linked by a methylene group (blue part in the figure below), limiting furan The flexibility of the ribose ring “locks” it into a rigid double-ring structure, but does not affect its compliance with the Watson-Crick base pairing principle, and can also hybridize with complementary DNA or RNA to form a duplex.
The synthesis of LNA is compatible with standard oligonucleotide synthesis, and single or multiple LNA nucleotide sites can be directly incorporated into the probe sequence selectively, and the recognition ability and Tm value of the probe can be adjusted.
The addition of LNA nucleotides can improve the thermal stability of the binding of the probe to the target sequence. For each LNA nucleotide added, the Tm value of the probe can be increased by 2~8°C, so the length of the probe can be designed to be shorter. GC content is also less dependent.
In the same sequence, the LNA-modified probe has a higher Tm value. The LNA-modified probe sequence can be designed to be shorter, which makes the LNA probe very suitable for the detection of shorter lengths or higher similarities. high target sequence.
LNA probes have high recognition ability and affinity can significantly improve the difference in Tm value between perfectly matched and mismatched bases and can more effectively distinguish SNPs.
LNA has high nuclease resistance, is relatively stable in vivo and in vitro, and can be applied to antisense technology.
Peptide Nucleic Acid Probes
Peptide Nucleic Acid (PNA) is a synthetic DNA analogue that retains the base and deoxyribose of DNA in structure, but the original ribose-phosphate backbone is replaced by an amide bond-like backbone of peptide bonds (red in the figure). Therefore, PNA still follows the principle of complementary base pairing, while the backbone is changed from the original negative charge to near neutrality, which weakens the electrostatic repulsion between double-stranded nucleic acids and makes the binding stability and specificity of LNA and DNA or RNA. are greatly improved. Moreover, the amide bond backbone of PNA is not easily hydrolyzed by nucleases and proteases, making it extremely stable both in vivo and in vitro.
The PNA probe has a higher Tm value. Compared with the conventional DNA-DNA sequence, each additional PNA base increases the Tm value by at least 1°C.
The electrically neutral backbone of PNA can reduce its electrostatic repulsion with DNA and RNA strands so that PNA has a higher affinity for the target sequence.
PNA has high specificity and high sensitivity, and the mismatch of one base pair can significantly reduce the Tm value, which can be used to distinguish single base mutations.
PNA has good stability and can exist stably under high temperature or high pH conditions; and because there is no recognition site for nucleases and proteases, it is more resistant to enzymatic degradation.
The hybridization and binding strength of PNA and the target sequence do not depend on the salt ion concentration, and the hybridization rate is faster, which can be increased by 100-5000 times.
The improvement of the probe, on the one hand, is to improve the detection signal-to-noise ratio, making the results more sensitive and specific; on the other hand, it is to enhance the flexibility of the probe design and make it more widely used.
The artificial transformation of standard nucleotides brings more possibilities, which not only greatly expands the application scope of oligonucleotide probes, but also opens new possibilities for artificially synthesized oligonucleotides. The application field can also make its application direction more controllable.
I&Y Biotech has a large-scale and high-throughput nucleic acid automatic synthesizer, preparative HPLC high-phase liquid chromatograph, LC-MS liquid chromatography-mass spectrometry analysis system, high-throughput capillary electrophoresis, freeze-drying equipment, molecular biology experimental platform (to verify the actual function of the primer-probe to verify the synthesis and purification process), etc. It can meet the requirements of both quality and quantity of probes in the field of qPCR.