Specific application of fluorescence in situ hybridization

Fluorescence in situ hybridization (FISH) is a physical mapping method, which uses fluorescein to label probes and detect hybridization between probes and metaphase chromosomes or interphase chromatin.

Fluorescence in situ hybridization (FISH) is a non-radioactive molecular cytogenetic technique developed on the basis of radioactive in situ hybridization in the late 1980s, and it is a new in situ hybridization method formed by replacing isotope labeling with fluorescent labeling. The probe first binds to the reporter molecule, and then connects with the fluorescent dye through immunocytochemical process. The basic principle of FISH is to label DNA (or RNA) probes with special nucleotide molecules, and then hybridize the probes directly to chromosomes or DNA fiber sections. Monoclonal antibodies coupled with fluorescein molecules specifically bind to probe molecules to qualitatively, locate and relatively quantitatively analyze DNA sequences on chromosomes or DNA fiber sections. FISH technology has the advantages of safety, rapidity, high sensitivity, long-term storage of probes, and multi-color display of metaphase and interphase nuclei. At the same time, multicolor fluorescence in situ hybridization and chromatin fiber fluorescence in situ hybridization were developed on the basis of fluorescence in situ hybridization.

For fluorescence in situ hybridization using rRNA, the following reasons will lead to lower fluorescence signal intensity:

Lower cell ribosome content

Low permeability around cells

The accessibility of the target sequence is low (due to the conformation generated by the folding of the rRNA, some positions in the rRNA molecule are in close contact with other chains or other rRNA or protein, so that the probe cannot hybridize with the target sequence).

In order to test whether the target sequence in the cell is easy to be hybridized by probes, and to test the optimal hybridization temperature, the experiment can be carried out by "clone -FISH": the rRNA gene is integrated into a plasmid, transformed into E.coli for expression, and then hybridized with a fluorescently labeled probe.

FISH can be combined with flow cytometry to count or separate specific fluorescently labeled cells.

Brief introduction of fluorescence in situ hybridization technology

1974, Evans combined chromosome banding technique with chromosome in situ hybridization for the first time, which improved the accuracy of localization. At the end of 1970s, people began to explore in situ hybridization with fluorescent markers, that is, FISH technology. 198 1 year, Harper successfully located a single copy of DNA sequence on G-banding specimens, which marked an important progress in chromosome mapping technology. In the 1990s, with the progress of the human genome project, FISH technology has been rapidly developed and widely used because of the need to draw high-resolution human genome maps.

1. principle

Fluorescence in situ hybridization is an important non-radioactive in situ hybridization technique. The basic principle is that if the target DNA on the detected chromosome or DNA fiber segment is homologous and complementary to the nucleic acid probe, a hybrid of the target DNA and the nucleic acid probe can be formed after denaturation-annealing-renaturation. When a nucleotide of a nucleic acid probe is labeled with a reporter such as biotin and digoxin, the DNA to be detected can be qualitatively, quantitatively or relatively located under a microscope by using the immunochemical reaction between the reporter and the specific avidin labeled with fluorescein.

2. Experimental process

Fish sample preparation → probe preparation → probe labeling → hybridization → (chromosome banding) → fluorescence microscope detection → result analysis.

3. Characteristics

According to the types of labeled molecules, in-situ hybridization probes can be divided into radioactive labels and non-radioactive labels. The advantage of isotope-labeled radioactive probe is that it has low requirements for sample preparation, and can enhance the signal intensity by prolonging the exposure time, so it is more sensitive. The disadvantages are unstable probe, long self-development time, low spatial resolution caused by radiation scattering and complicated isotope operation. Using fluorescent labeling system can overcome these shortcomings, which is FISH technology. As a non-radioactive detection system, FISH technology has the following advantages: 1, fluorescent reagents and probes are economical and safe; 2. The probe is stable and can be used within two years after once labeling; 3, the experimental period is short, the results can be obtained quickly, the specificity is good, and the positioning is accurate; 4.FISH can locate the DNA sequence with the length of 1kb, and its sensitivity is equivalent to that of radioactive probes; 5. Multicolor FISH can detect multiple sequences at the same time by displaying different colors in the same core; 6. The change of metaphase chromosome number or structure can be displayed on the glass slide, and the structure of interphase chromosome DNA can also be displayed in suspension.

Disadvantages: 100% hybridization can not be achieved, especially when short cDNA probes are used, the efficiency is obviously reduced.

4. Application

This technique can be used not only for chromosome location of known genes or sequences, but also for cloning genes or genetic markers and the study of chromosome aberration. It has advantages in the research of gene qualitative, quantitative, integration and expression.

In this section, edit the development history of fluorescence in situ hybridization.

1 FISH technology to detect the number of websites and develop detection targets.

After the basic establishment of FISH technology, FISH technology is not only used to detect a single gene or nucleic acid, but also extended to the simultaneous detection of multiple gene loci in polychromatic fish, from gene detection to in-situ detection of transcripts in genome, chromosome and living cells and nucleic acid detection at the tissue level, and mRNAs will be applied to the detection of the whole organism in future research. The early probes were very large, and specific hybrid clones were obtained through vector proliferation, gap translation, in vitro transcription and random primer DNA synthesis. However, large fragments of probes usually have repetitive sequences, resulting in high fluorescence background. The pretreatment method of inhibiting nonspecific hybridization with unlabeled nucleic acid combined with nonspecific sites can overcome the above problems, and researchers can expand the detection target and realize whole chromosome staining. In cytogenetics, FISH technology has significantly improved in chromosome analysis. For example, comparative genomic hybridization is used to detect deletion and replication of chromosome regions. Once the large fragment probe is nonspecific bound to the sample, it will form a signal, which confuses the detection of genes on the chromosome and needs to be cut into small fragments of < 200 nucleotides. Nowadays, the improvement of detection means and the continuous development of detection software make the detection requirements of FISH technology lower and lower, and the sensitivity is higher and higher. With the continuous improvement of accurate computer image processing algorithm, the high resolution technology of probe at submicroscopic level has been formed. With the detection target getting smaller and smaller, FISH technology has been used for hidden sub-telomere karyotype gene rearrangement, accurate chromosome location and single copy mRNA detection.

With the expansion of FISH detection range, the application of FISH technology increased rapidly in 1990s. The branch technology formed by FISH technology has realized the simultaneous detection of more and more different types of loci. First, different fluoresceins are used to detect multiple sites, such as two-color fluorescence to detect specific nucleic acid sequences, and each chromosome, gene or transcript is represented by a distinguishable fluorescence signal. After that, two color coding schemes were adopted, which further expanded the application scope of FISH. The decoding scheme mainly describes the multi-site according to the color ratio, that is, the proportion of each color in the total color. Each of the above methods, or the combination of the two methods, detected as many as 12 loci. The five-color scheme of computer translation can detect all human chromosomes at the same time, which represents a milestone in multi-site detection of fish technology. Although mRNAs can be observed by many methods, it seems to be more promising to analyze the whole transcript in situ by FISH. Color coding technology realizes the detection of the whole organization.

2 FISH technology in quantitative analysis stage

Pinkel et al. (1986) used the quantitative analysis of fluorescence images for basic cytogenetics detection for the first time, and used a two-color excitation block device camera to detect fluorescence signals, and the quantitative analysis technology was quickly used for mRNA detection. The key of fluorescence detection is the reproducibility and irregularity of signal and the autofluorescence of background. Not only the fluorescence of different samples is different, but also the material on the same slide or the same cell may have uneven fluorescence. At present, there are many ways to eliminate autofluorescence in some tissues: for example, in the process of sample preparation, sodium borohydride is used as a reagent to eliminate autofluorescence, or pretreatment with illumination radiation is used to eliminate nonspecific background signals. These methods of eliminating autofluorescence are not completely effective, and the autofluorescence signal is usually eliminated by computer operation in image analysis. The spectral data of fluorescence image includes real signal and many noises, which are analyzed separately and removed by separate spectral component analysis. Multicolor FISH has its own limitations, including different fluorescence intensity and color overlap. However, multi-color images are balanced by computer algorithm analysis, including automatic correction of intensity change and signal overlap.

The limitation of fish image itself has not affected the development of automatic decoding algorithm. DNA locus detection with large sequence probes and multicolor fluorescence counting algorithm assist pathologists to realize automatic analysis. In addition, the application of detection probe kit and counting method provides a platform for convenient detection results. Although many methods have been used to analyze or optimize the automatic cell detection system, manual cytopathological detection is still a highly reliable tissue analysis method. However, in the future medical diagnosis, the high efficiency of cell preparation, identification and computerized detection of cell samples on fixed media can not be ignored, and the rapid detection of intracellular molecular signals can only be detected by computer-aided methods. Now, the automatic detection program has been extended to use polygene transcription model to detect specific DNA clusters and transcription sites to determine the state of functional cells.

Development of 3 FISH technology in detection field

Because the initial development of FISH is mainly the expansion of probe types and detection sites, the future development of fluorescence detection technology may include the expansion of detection fields. The clinical diagnostic application of fluorescence images needs to be further improved in the detection system, such as the combination of probes, the automation of photography and analysis, so as to avoid errors between different operations. Sample thickness is a limiting factor in detecting sample types by fluorescence microscope. The recent laser confocal microscope and optical X-ray tomography technology require the sample thickness to reach 1~2mm. An improved optical projection X-ray microscopy technique can obtain the sample image with the thickness of 65438±0.5mm, which expands the detection range of biological and diagnostic samples. The detection of living cells by RNAs FISH technology is also reported. Both fluorescent groups released in vivo and fluorescein from hybridization probes can be used for detection. These two new methods can reduce the high background (such as living cells) in the presence of nonspecific labeled probes, and can be used to track the synthesis and metastasis of mRNA. These methods are easier to detect different target molecules than green fluorescent protein (GFP). Compared with in situ hybridization detection of GFP living cells, FISH is easily influenced by intracellular synthetic probes. FISH needs to be further improved to reduce the interference background of gene expression detection in vivo and avoid the interference of intracellular self-hybridization. In fact, there is no need to consider the difference between FISH detection and fluorescent protein detection. The combination of FISH technology and fluorescent protein technology can detect target nucleic acid and protein at the same time.

The application of multiphoton microscopy has further expanded the application scope of fluorescence images. Using a multiphoton microscope, the laser block can emit photons and focus them on the microscope to excite the target fluorescein twice or three times. Near-infrared excitation light can penetrate deeper into biological samples and is less toxic to living samples than visible light. The application of this new method has applied fluorescent images to detect life systems, even the whole animal body. Because it is impossible to synthesize biomarker probes, the application of fluorescence images in vivo is limited to detecting the autofluorescence of fluorescent molecules or organisms. Autofluorescence signals generated by biological tissues in normal physiological or pathophysiological processes can be used as important diagnostic signals. Once the biological probe is possible, it will become a powerful auxiliary means to identify specific nucleic acid sequences, make non-invasive diagnosis and obtain diagnostic images.