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Fluorescence in Situ Hybridization

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Fluorescence in situ hybridization can be used to detect specific DNA or RNA in cells to determine the expression and location of specific genes. It can also be used to detect chromosomal changes in tumors or other diseases.

Fluorescence in situ hybridization

FISH (fluorescence in situ hybridization) technology is a new positioning technology developed in the 1980s. It has been widely used in human genome research. For the identification of syngeneic clones, gene mapping can be performed at a resolution of 50 kb through FISH of interphase nuclei. The latest research progress has been able to perform FISH of decomposed chromatin fiber to directly measure the length of the gene. The purpose of advanced genetic mapping. Through the development of FISH technology, it plays a role in human and other types of genome research.

Fluorescently labeled chromosome in situ hybridization technology provides a fast and effective means to link DNA fragments with specific eukaryotic cell chromosomal zones, and to sequence these DNA fragments. This is the study of DNA sequence on chromosomes. The most direct way to get to the position.

Two basic methods can be used for probe labeling:
(1) Direct labeling method: label the fluorescent molecules directly on the probe DNA/RNA, and then directly detect under a fluorescence microscope after crossover. This method is fast and simple. Because the crossover signal is weak and cannot be further amplified, this method was not used much before, but it has the advantage of less background. Recently, it has been applied in the kits of some companies.
(2) Indirect labeling method: The commonly used indirect method is to incorporate some recombinant hapten (hapten) labeled molecules into the probe molecules. Alternative reagents used are biotin, digoxigenin, dinitrophenyl (DNP), aminoacetylfuorene (AAF), mercury and sulfonate. Now more people start to use random primer method. The size of the probe labeled with these two methods is in the range of 200-500bp, which is the best size for crossover. In addition, it can also be extended by PCR between two primers of a known sequence, or be transcribed from a suitable vector by RNA.

1. principle

Its basic principle is: use a known nucleic acid sequence as a probe, directly labeled with fluorescein or labeled with a non-radioactive substance and hybridize with the target DNA, and then connect the fluorescein label through the immunocytochemical process, and finally in the fluorescence microscope Observe the crossover signal to perform quantitative, qualitative and localization analysis of the nucleic acid to be tested.

2. experiment process

FISH experiment process
FISH experiment process

FISH sample preparation→probe preparation→probe labeling→hybridization→chromosome banding→fluorescence microscope inspection→result analysis.

3. Features

The advantage of using isotope-labeled probes is that the requirements for sample preparation are not high, and the signal intensity can be increased by extending the exposure time, so it is more sensitive. The disadvantages are the probe aperture, long auto-radiography time, low radiation scattering and spatial resolution, and cumbersome isotope operations. Using fluorescent labeling system can overcome these shortcomings, this is FISH technology. , Has the following advantages:
First of all, from the perspective of probe preparation crossover, there is no duplication of biotin and other labeling molecules, and the labeling process and crossover process have no risk of contamination and are relatively safe. Moreover, the probe molecule after labeling with biotin or other labeling molecules is quite stable, has no half-life limitation, and can be stored for a long time. Fluorescence color development time is short, does not require repeated exposure like scattering cross, and the background is simple. The sensitivity is not inferior.
In addition, FISH can use multiple colors to develop colors at the same time, which is incomparable to isotope crossover. This kind of multi-color mapping enables simultaneous observation of the chromosome banding and the prominent color of the probe signal. Different probes can also be labeled with different fluorescence, and the relative positions ofA the probes on the chromosomes can be determined.
Disadvantages: 100% crossover cannot be achieved, especially in the application of promising cDNA probes, the efficiency is significantly reduced.

4. Application

This technology can be used not only for chromosomal location of known genes or sequences, but also for the study of uncloned genes or genetic markers and chromosomal aberrations. It has advantages in the research of gene qualitative, quantitative, integration and expression.
I. Detection of chromosome structural variation and aneuploidy: fluorescence in situ crossover simplifies the detection of chromosome structural variation. In situ crossover can be used to easily detect missing, added or replaced chromosomes.
II. Measurement of gene amplification and deletion: The spatial resolution and sensitivity of FISH are made possible by the localization of parental and amplified genes in disease- and insect-resistant cells. FISH technology can also be used to detect some gene deletions related to genetic diseases, such as the successful detection of aniridia disease (a rare genetic abnormality with missing iris).
III. FISH technology provides an important means for the study of centromere structure. At the same time, the application of FISH technology can directly observe chromosome telomeres, which simplifies the study of the structure and function of chromosomes in the nucleus.
IV. Gene mapping: FISH technology can directly detect the position of DNA on the chromosome, and the determined position is the actual physical location of the gene on the chromosome. Due to the influence of in-situ crossover without shifting intra-site variation and inter-site copy number, FISH technology has become an important method for mapping repetitive sequences and multi-gene families.
V. Chromosomal RNA and genome evolution research: The main components of chromosomes include DNA and histones, in addition, there are non-histones and RNA. Precise positioning of the distribution of these substances in chromosomes is to study the high-level structure of chromosomes and replicate chromosome FISH technology provides an effective means for the above research.

Fluorescence in-situ crossover technology mainly replaces the following areas:
·Analysis of cell transcription profile
·In vivo and in vitro RNAi delivery and gene knockout
·Biomarker research
·Reporter gene screening
·Molecular pathology
·Stem cell differentiation
·Cell Biology

With the development of labeling methods, detection reagents, and fluorescence observation, the application range of chromosome FISH is expanding and the development of laser coexistence focusing technology, so that the three-dimensional reconstruction of the nucleus can be achieved with the aid of computers. It can be used to locate genes and to study the position of genes in the internucleus.

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