In the late 1980s, fluorescence in situ hybridization (FISH)In situ hybridizationA non radioactive substance developed on the basis ofmolecular biologyandcytogeneticsThe combined new technology is replaced by fluorescent labelingIsotope labelingA new method of in situ hybridization was developed.[1]
In 1977, fluorescent labeled antibodies were used to identify specific DNA RNA hybridization II.
In 1980, J.G. Baunlan et al. combined fluorescein withRNA probeIt is used for direct and rapid detection of specific target sequences.[2]
Technical Principles
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The principle of fluorescence in situ hybridization technology is tofluoresceinDirectly or indirectly markedNucleic acid probe[or nucleic acid probes labeled with biotin, digoxin, dinit prophenyl (I) NP), aminoacetyl AAF fluorine (AAF), etc. and nucleic acid sequences in the samples to be tested are in accordance withcomplementary base pairing After washing directly in thefluorescence microscopeLower observation.[2]
Fluorescence in situ hybridization (FISH) is an important non radioactive in situ hybridization technology. The principle is to use reporter molecules (such as biotin, digoxin, etc.) to label nucleic acid probes, and then combine the probes with targets on chromosomes or DNA fiber slicesDNA hybridizationIf the two are homologous and complementary, a hybrid of target DNA and nucleic acid probe can be formed.At this time, the immunochemical reaction between the report molecule and the specific avidin labeled with fluorescein can be used to conduct qualitative, quantitative or relative positioning analysis of DNA under the microscope through the fluorescence detection system.[1]
Technical advantages
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And othersIn situ hybridizationCompared with FISH, FISH has many advantages, mainly reflected in:
① Not required by FISHradio isotopeMarking is more economical and safe.
② FISH has the advantages of short experimental cycle, high stability, good specificity, accurate positioning and rapid results.
③ FISH enhances the hybridization signal and improves the sensitivity through multiple immunochemical reactions, and its sensitivity is equivalent to that of radioactive probes.
④ Multicolor FISH can simultaneously detect multiple sequences by displaying different colors in the same core.
⑤ The number or structure of metaphase chromosomes can be displayed on the slide.Intervals can also be displayed in suspensionChromosome DNAStructure of.[1]
technological development
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（1） Multicolor fluorescence in situ hybridization (mFISH)
MFISH is a new technology developed on the basis of fluorescence in situ hybridization. It not only has the advantages of FISH, but also overcomes many limitations of FISH. Its biggest feature is that it can complete multiple FISH experiments and a variety of different gene locations in one FISH experiment.MFISH can simultaneously detect multiple genes and distinguish complexChromosome translocationIt can distinguish polyploid and superdiploid of interphase cells.MFISH uses fluorescence bands with different excitation and absorption spectra to label different probes according to certain color matching methods, so that different target DNA can be located and analyzed simultaneously, and the positions of different probes on chromosomes can be sorted.
The methods of probe fluorescein color matching include non color matching, mixed color matching and proportional color matching.Among the three methods, the proportional method requires only a few fluorescein to label a variety of probes, so it has more potential for development.Chromosome painting, comparative genomic hybridization (CCH), spectral karyotyping,Mulicolor PRINS) and other technologies are developed on the basis of mFISH.
（2） DNA fiber FISH
The resolution of FISH depends on the concentration of carrier DNA. How to improve the resolution has always been an important issue.Wiegant et al. and Heng et al. first linearized the chromosome by chemical method, and then used it as a carrier for FISH to significantly improve its resolution, which is the original fiber FISH.Fiber FISH uses various technologies to prepare DNA fiber from all genetic material of the cell to be studied, that is, DNA, on the slide, and hybridizes with probes labeled with fluorescent substances of different colors with DNA fiber. The results are observed and analyzed under the fluorescence microscope.
The key of fiber FISH is to prepare high-quality linear DNA fiber.Ideally, the length of the prepared DNA should be close to that of the fully naturally stretched DNA fiber, and the breaking points should be as few as possible.In recent years, many methods for preparing DNA fibers have been developed.Fiber FISH can be used for quantitative analysis. It requires less templates and has the advantages of high resolution and sensitivity.Therefore, fiber FISH plays a very important role in chromosome mapping, gene recombination research and clinical chromosome gene sequence detection.[1]
Technology application
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As a molecular cytogenetic technique for visualizing specific DNA sequences, fluorescence in situ hybridization (FISH) is widely used in chromosome aberration.Such as aneuploid, chromosome recombination.Its basic process includes probe labeling, probe denaturation, sample denaturation, hybridization and fluorescence signal acquisition.
Fluorescence in situ hybridization (FISH) technology has considerable advantages in gene qualitative, quantitative, integration, expression and other aspects of research. At present, it has been widely used in many fields such as genetic disease diagnosis, virus infection analysis, prenatal diagnosis, tumor genetics and genome research, and plays an important role in clinical testing, teaching and research.
（1） Chromosome location and mapping of genes (or DNA fragments)
At present, the main method of gene mapping is FISH.The isolated DNA sequence can quickly determine the mutual order and distance between a series of DNA sequences and complete gene mapping by directly passing through FISH, using a variety of color fluorescein labeled probes, and combining information on metaphase chromosomes and interphase cells.When two different DNA strands are labeled with different colors of inflammatory light cords, and their distance on the chromosome is greater than 1Mbp, their order on the chromosome can be distinguished according to the arrangement relationship of different probe signals.
If the cells are treated with 5-bromodeoxyuracil (5-Burd), the chromosomes with high resolution banding can be obtained, and the sorting ability of DNA chain labeling to chromosomes can be improved.If interphase cells are used, the distance between two DNA strands can be shortened to 50kb, which is 1/20 of the resolution distance on chromosomes. The order of different probes can be determined by measuring the distance between interphase cells.The determination of the fine position of DNA strands on chromosomes is applicable to the detection of some special chromosomal translocations and deletions.By marking the same DNA chain and hybridizing chromosomes of different species and genera, we can find homologous genes between different species and genera and their positions on chromosomes, so as to understand the evolutionary relationship between species and genera.
（2） Abnormal chromosome number and structure
In cytogenetics, probes for repetitive sequences are most widely used, including a-satellite DNA probes, beta satellite DNA probes and classical satellite DNA (elasic - stllite DNA) probes.A SatelliteDNA probeThe centromere of human chromosome was mainly detected.The β satellite DNA probe is located around the acrocentric chromosome and the heterochromatin of the chromosome.Classic satellite DNA probes have AATCG short segment repeats located on chromosomes 1, 9, 15, 16 andY chromosomeLong arm heterochromatin around.The latter two probes can be used not only for chromosome number inspection, but also for the inspection of fine changes in the above parts.applicationFISH technologyDetection of chromosome number and structural abnormalities has high specificity and sensitivity, and has been widely used in rapid prenatal diagnosis.
The FISH detection of blood tumors in clinic mainly focuses on the detection of fusion genes formed by heterotopic chromosomes, such as ber/abl translocation DNA probe, t (15;17) Translocation DNA probe and t (18; 21) translocation DNA probe;Gene deletion detection can find the deletion of some key genes, which is helpful for disease diagnosis and prognosis judgment;Fluorescence in situ hybridization can be used to detect small residual lesions, andhematopoietic stem cell transplantationStatus monitoring.
（4） Solid Oncology
Before FISH technology, all the methods used to measure gene amplification were classic molecular biological methods. Compared with FISH, these methods not only took time and effort, but also could not observe the state of gene amplification at the cell level.The greater advantage of FISH technology is that direct evidence of DNA amplification can be observed on interphase nuclei, and the amount and intensity of fluorescence signals of amplified DNA displayed by interphase nuclei are often related to the level of DNA amplification.
FISH is widely used in the auxiliary diagnosis of solid tumors, such as breast cancer, bladder cancer, cervical cancer, lung cancer and lymphatic cancer.The amplification of Her-2/neu gene in breast cancer cells often indicates poor prognosis of patients, and 25%~30% of breast cancer patients have Her-2/neu gene amplification and/or overexpression.Using Her-2/neu gene DNA probe to detect the amplification and expression level of Her-2/neu gene is beneficial to breast cancerclinical diagnosisAnd efficacy monitoring.Chromosome centromere specific probe can be used to analyze chromosome number variation of interphase cells. For example, Hopman used FISH technology to study bladder cancer and found the loss of chromosome 9.A variety of chromosome probes labeled with different colors can be used to study the heterogeneity of tumor chromosome number changes.At present, FISH is mainly used for early diagnosis, efficacy testing, individualized treatment and prognosis judgment of tumors.[1]
