It has characteristic fluorescence in the ultraviolet-visible-near-infrared region, and its fluorescence properties (excitation and emission wavelengths, intensity, lifetime, polarization, etc.) can be sensitively changed with the properties of the environment, such as polarity, refractive index, viscosity, etc. a class of fluorescent molecules.
After being excited by the excitation light, it returns to the ground state from the excited state singlet state, and has characteristic light emission in the ultraviolet-visible-near-infrared region, which is called fluorescence. A class of fluorescent molecules whose fluorescence properties (excitation and emission wavelengths, intensity, lifetime, polarization, etc.) can be sensitively changed with the properties of the environment, such as polarity, refractive index, viscosity, etc., are called fluorescent probes. There are many types of fluorescent probes, which can be divided into organic and inorganic probes according to material properties, molecular probes and nanoprobes according to the size of the probes, and single-photon, two-photon and multi-photon fluorescent probes according to the excitation light source. The needles can also be classified into metal ion fluorescent probes and biomolecular fluorescent probes according to the analyte. Fluorescent probes are widely used in various detection and labeling, such as determination of metal ions, pesticide residues, biomolecule content, trace biomolecules, labeling of macromolecules and cellular and subcellular structures.
The fluorescent chemicals used in real-time PCR can be divided into two types: fluorescent probes and fluorescent dyes. During PCR amplification, a specific fluorescent probe is added along with a pair of primers. The probe is an oligonucleotide, and the two ends are respectively labeled with a reporter fluorescent group and a quenching fluorescent group. When the probe is intact, the fluorescent signal emitted by the reporter group is absorbed by the quencher group; during PCR amplification, the 5′-3′ exonuclease activity of Taq enzyme cleaves and degrades the probe, making the reporter fluorophore and the quencher group. The fluorescent group is separated, so that the fluorescence monitoring system can receive the fluorescent signal, that is, a fluorescent molecule is formed every time a DNA chain is amplified, and the accumulation of the fluorescent signal is completely synchronized with the formation of the PCR product.
Determination of Target Genes and Design Principles of Primers and Probes by Real-time Fluorescent PCR Technology
1. Determination of target genes: select genes common to the detection group to avoid false negatives (missed detections).
2. Determination of detection fragments: select the conservative (less mutation) within the detection group (to avoid false negatives), and the specific sequences between groups (with more mutations) (to avoid false positives) as the design position of the primer probe. Fragment length 70-150bp.
3. Primers: length 17-25bp; GC content 30-80%; annealing temperature (Tm value) 58-60 ℃; avoid stable primer-dimers (especially multiplex detection) and hairpin structures (free energy greater than – 3.5kc/m); the sequence cannot appear continuous G, avoid G or C at the 3′ end, and avoid more than two G or C within the last 5 bases.
4. Probe: length 20-30bp (Taqman) or 16-25bp (MGB); GC content 30-80%; Tm value 68-70 ℃; avoid stable dimer and hairpin structure (free energy greater than – 3.5kc/m); the 5′ end cannot be G; the position should be as close to the upstream primer as possible; the fluorescent label with large wavelength difference (multiplex detection) or Fam (single detection) should be selected.
Function and use
It is most commonly used to label antigens or antibodies in fluorescent immunoassays, and can also be used to detect microscopic properties in microenvironments, such as surfactant micelles, bimolecular membranes, and protein active sites. It is usually required that the probe has a large molar absorption coefficient and a high fluorescence quantum yield; the fluorescence emission wavelength is at a long wavelength and has a large Stokes shift; when used in immunoassays, the binding to antigens or antibodies should not affect their activity. .
It can also be used to label undetermined nucleotide fragments, and to specifically and quantitatively detect the amount of nucleic acid.
Commonly used fluorescent probes include fluorescein-based probes, inorganic ion fluorescent probes, fluorescent quantum dots, molecular beacons, and the like. In addition to the quantitative analysis of nucleic acids and proteins, fluorescent probes are widely used in nucleic acid staining, DNA electrophoresis, nucleic acid molecular hybridization, quantitative PCR technology and DNA sequencing.
Currently, methods for detecting fluorescent probes mainly include single-point assay and charge-coupled device (CCD) fluorescence imaging (including confocal fluorescence microscopy imaging for micro-area analysis). Due to the long scanning time of the photomultiplier tube and the high laser irradiation intensity, it is difficult to capture the early changes of fluorescence. The CCD fluorescence imaging has a large area array, a wide imaging field, and an adjustable imaging time, so the detection effect is better.
The biggest feature of chemiluminescence detection is simple equipment, simple operation, fast analysis speed and high sensitivity. Chemiluminescence imaging analysis (CLI) is a combination of chemiluminescence and imaging technology, which has the characteristics of high resolution, simultaneous detection of multiple samples, wide spectral response range and high sensitivity. It has been widely used in gels, protein Chemiluminescent signal detection in imprinting and microarray chips. In this experiment. Since chemiluminescence does not require any light source, when performing chemiluminescence imaging detection on fluorescent probes, there is no interference of fluorescence detection or unavoidable optical background during fluorescence imaging, so that a lower detection limit can be obtained. Five kinds of fluorescent probes were quantitatively analyzed by this system, and the chemiluminescence imaging of monoclonal goat anti-human IgG labeled with tetramethylisothiocyanate rhodamine (TRITC) was studied, and the detection limit reached 10-11mol/ L, an order of magnitude lower than the detection limit of fluorescence imaging under the same conditions.