What are the fluorescent probes for pcr

I. TaqMan probe

1. Structure

TaqMan probe is a double-labeled, self-quenching hydrolysis probe, and its 5 end is labeled with fluorophore (such as FAMan probe), HEX, etc.). ", 3 ends are marked with quenching groups (such as TAMRA, BHQ, etc.)."

Among them, the development of quencher has undergone some selective evolution. TAMRA, the earliest quender, was used with FAM fluorophores to achieve the purpose of real-time fluorescence detection. However, TAMRA, as a quenching agent with fluorescence, has a high background signal and a narrow wavelength selection range, and its application is very limited.

Later, Dark Quenchers, such as DABCYL, were developed. Such quenches emit no light in the visible spectrum, unlike fluorescence quenches such as TAMRA, and the excitation energy absorbed from the fluorophore is consumed as thermal rather than optical energy, thereby reducing the background signal. But DABCYL can only absorb wavelengths in a small range, which limits its application in multiple QPCRS.

To meet multiple qpcr applications, the Black Hole Quencher (BHQ) poop was developed. BHQ can operate at longer wavelengths and has an efficient quenching capability in the visible spectrum, matching a wide selection of fluorophore wavelengths. The quenching wavelength range of the existing BHQ dyes can be considered from 430 nm to 730 nm, and multiple QPCR assays can be achieved by using different BHQ and different fluorophores. In order to avoid patents or improve the quenching effect, some companies have developed other similar quencher, such as IDT(Integrated DNA Technologies) company quencher group: IBFQ(Iowa Black®️ FQ) and IBRQ(Iowa Black)

RQ)。

Step 2: Principles

Based on the principle of real-time QPCR(Fluorescence Resonance Energy Transfer, FRET) assay, that is, when the probe is in the intact state and the fluorophore is relatively close to the quenching group due to folding and curling, the quenching group can absorb the fluorescence emitted by the fluorophore. The guiding instrument could not detect the signal; When the probe binds to the target sequence and is hydrolyzed by Taq enzyme during extension, its fluorophore is released into the reaction system, separated from the quenched group, and the instrument can detect the signal generated under the excitation light.

Ii. MGB probe

1. Structure

In addition to the fluorescent group and the quenching group, the MGB probe is labeled with 3 '5' probe 'Minor groove binding (MGB) at each end, wherein the labeled quenching group is the non-fluorescent quencher (NFQ), and the labeled quenching group is the non-fluorescent quencher (NFQ). MGB is mostly a small-molecule tripeptide that can noncovalently bind to double-stranded DNA minor grooves and stabilize the DNA structure.

MGB probes included MGB-taqman probes. Mgb-three types: Pleiades probe and MGB-Eclipse probe. The Taqman-MGB probe is the MGB attached to the 3 end of the TaqMan probe, which acts as a hydrolytic probe to release the fluorescent group to generate the signal after being hydrolyzed by Taq enzyme during the extension process. Pleiades- A fluorophore was labeled at the 5 end of the MGB probe and a quenching group was connected to the MGB and 3 ends. The Eclipse-MGB probe was labeled with the quench group at the 5 end, and the MGB was connected, and the fluorophore was labeled at the 3 end. Both the Pleiades-MGB probe and the Eclipse-MGB probe are hybridization probes, and the MGB attached to the 5 terminus prevents Taq enzyme hydrolysis, so that the fluorogenic and quenched groups generate fluorescent signals after hybridization to the target sequence.

Step 2: Advantages

The TM value of the MGB modified probe combined with the target sequence can be increased by 15~30℃, which makes the designed probe sequence shorter.

The non-fluorescence quenching group (NFQ) was used in the MGB probe, which could greatly eliminate the background fluorescence produced by the traditional quenching group and improve the signal-to-noise ratio.

MGB combined with DNA double-stranded minor groove can stabilize AT-rich sequences and reduce the influence of TM value on the experiment.

MGB probes can be used to detect conserved sequences or SNPS in short fragments. The mutation site was designed in the middle third of the probe, which could be used to distinguish single base mutations.

The expanded product can also be subjected to melting curve analysis after detection with Pleiades-MGB probes or Eclipse-MGB probes.

Three, double quenched probe

1. Structure

The traditional single-quenched probe 5 has a fluorophore at the end and a quench group at the end of 3, while the double-quenched probe adds a second quench group within the probe to make the quenching effect more complete.

In the case of the IDT double-quenched probe, the second quench group on the base junction at position 9 or 10 of the probe: ZEN™️ quench or TAO™️ quench.

"ZEN double-quench probes contain 5 '-terminal fluorophores, a 3' -terminal Iowa Black™ FQ(IBFQ) quench group, and proprietary built-in ZEN quench group. The most commonly used 5 'FAMM includes terminal fluorophores including FAM, TET, Yakima Yellow, SUN, or HEX."

TAO double-quenched probe 5 '-end Cy 5 fluorophore, a 3' -end Iowa Black RQ (IBRQ) quench group, and proprietary built-in TAO quench group.

Step 2: Advantages

Double-quenched probes have a double guarantee. In addition to the ordinary quench group at the 3 end of the probe, a second quench group was added to reduce the bottom signal and improve the signal-to-noise ratio.

The traditional probe length is generally 20-30 bp, while the double-quenched probe can be designed to be longer, up to 40 bases.

In multiple qpcr assays, double-quenched probes can reduce the crosstalk of fluorescence signals in multi-channel assays.

The double-quenched probe also enhances the stability of the double-stranded structure, prevents enzymatic cleavage by exonuclease, is non-cytotoxic, and can be applied to anti-miRNA sequences and miRNA, in situ hybridization, such as MRNA.

4. Locked nucleic acid probe

1. Structure

Locked nucleic acid (LNA) is a high-affinity RNA analog that is linked by a methylene group (blue below), which limits the flexibility of the furan ribose ring, "locking" it into a rigid double-loop structure, but does not prevent it from adhering to the Watson-Crick principle of base pairing. It can also hybridize with complementary DNA or RNA to form a duplex.

Step 2: Advantages

LNA synthesis is compatible with standard oligonucleotide synthesis. Single or multiple synthetic Lnas can directly mix nucleotide sites selectively with probe sequences to adjust probe recognition ability and TM value.

The addition of LNA nucleotides improves the thermostability of probe binding to the target sequence. The TM value of the probe can be increased by 2-8 ℃ with each addition of LNA nucleotide, and the probe length can be designed to be shorter and the GC content of the dependent sequence is lower.

For the same sequence, the TM value of the probe modified by LNA was higher. For the same TM value, the LNA modified probe sequence can be designed to be shorter, making the LNA probe very suitable for detecting short length or high similarity target sequences.

LNA probes have high recognition ability and affinity, which can significantly improve the TM value difference between perfectly matched and mismatched bases, and distinguish SNPS more effectively.

LNA has high nuclease resistance and is relatively stable in vitro and in vivo, which can be applied to antisense technology.

V. Peptide nucleic acid probe

1. Structure

Peptide Nucleic Acid (PNA) is a synthetic analog of DNA that structurally retains the bases and deoxyribose of DNA, but the original ribose phosphate skeleton is replaced by an amide bond skeleton that resembles peptide bonds (marked by red circles). Thus, the PNA still follows the principle of complementary base pairing, while the skeleton changes from the original negative to nearly neutral, weakening the electrostatic repulsion between double-stranded nucleic acids and greatly improving the binding stability and specificity of LNA to DNA or RNA. Moreover, the amide bond backbone of PNA is not easily hydrolyzed by nucleases and proteases, making it extremely stable in vitro and in vivo.

Step 2: Advantages

Compared with the traditional DNA-DNA sequence, the PNA probe has a higher TM value, and the TM value increases by at least 1℃ for every additional PNA base.

The electrically neutral backbone of PNA can reduce electrostatic repulsion from DNA and RNA strands, resulting in higher affinity of PNA to target sequences.

PNA has high specificity and sensitivity, and the base pair mismatch can significantly reduce the Tm value, which can be used to distinguish single base mutations.

PNA has good stability and can exist stably at high temperature or pH. Because there is no recognition point for nucleases and proteases, the resistance to enzymatic degradation is also stronger.

The hybridization binding strength of PNA to the target sequence is independent of the salt ion concentration, and the hybridization rate can be increased by 100 to 5000 times.

In conclusion, on the one hand, the improvement of probes is to improve the detection signal-to-noise ratio and make the results more sensitive and special. The other is to improve the flexibility of probe design and make it more widely used. Manual modification of standard nucleotides brings more possibilities, which can not only greatly expand the application range of oligonucleotide probes, but also open up new application fields for synthetic oligonucleotides and make their application direction more controllable.