Fluorescence in situ hybridization (FISH) is a new technology that combines non-radioactive molecular biology and cytogenetics developed on the basis of radioactive in situ hybridization in the late 1980s. A new in situ hybridization method based on isotope labeling.
Fluorescence in situ hybridization was introduced in the late 1970s.
In 1977, fluorescently labeled antibodies were used to recognize specific DNA-RNA hybrids.
In 1980, J.G.Baunlan et al. applied chemical coupling to combine fluorescein to RNA probes for direct and rapid detection of specific target sequences.
The principle of fluorescence in situ hybridization technology is to directly or indirectly label nucleic acid probes with fluorescein [or biotin, digoxigenin, dinit rophenyl (I) NP), aminoacetylAAFfluorine (AAF) and other labeled nucleic acid probes with the sample to be tested. The nucleic acid sequence in the hybridization was carried out according to the principle of complementary base pairing, and after washing, it was directly observed under a fluorescence microscope.
Fluorescence in situ hybridization is an important non-radioactive in situ hybridization technology. The principle is to label nucleic acid probes with reporter molecules (such as biotin, digoxigenin, etc.) DNA hybridization, if 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 reporter molecule and the specific avidin labeled with fluorescein can be used to conduct qualitative, quantitative or relative localization analysis of the DNA under a microscope through a fluorescence detection system.
Compared with other in situ hybridization techniques, fluorescence in situ hybridization has many advantages, mainly reflected in:
①FISH does not require radioisotope labeling, which is more economical and safe.
②The experimental period of FISH is short, the probe has high stability, good specificity, accurate positioning, and results can be obtained quickly.
③FISH enhances the hybridization signal and improves the sensitivity through multiple immunochemical reactions, and its sensitivity is comparable to that of radioactive probes.
④Multicolor FISH can detect multiple sequences simultaneously by displaying different colors in the same nucleus.
⑤ Changes in the number or structure of chromosomes in metaphase can be displayed on glass slides. The structure of interphase chromosomal DNA can also be visualized in suspension.
(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. done in one FISH experiment. mFISH can detect multiple genes at the same time, distinguish complex chromosomal translocations and small deletions, and distinguish between polyploidy and hyperdiploidy of interphase cells. mFISH uses fluorescent cords with different excitation and absorption spectra to label different probes according to a certain color matching method, so that different target DNAs can be located and analyzed at the same time, and the positions of different probes on the chromosome can be sorted.
The color matching methods of probe fluorescein include non-tinting method, mixed coloring method and proportional coloring method. Among these three toning methods, the proportional toning method can label a variety of probes with only a few fluorescein, so it has more potential for development. Chromosome painting, comparative genomic hybridization (CCH), spectral karyolyping (SKY), cross-species colorbanding (Rx-FISH) and multiple Technologies such as mulicolor primed in situ labeling (mulicolor PRINS) are developed on the basis of mFISH.
(2) DNA fiber fluorescence in situ hybridization (DNA fiber-FISH)
The resolution of FISH depends on the degree of enrichment of the vector DNA, and how to improve the resolution has always been an important topic. Wiegant et al. and Heng et al. first used chemical methods to linearize the chromosome, and then used it as a carrier for FISH to significantly improve the resolution, which is the original fiber-FISH. Fiber-FISH uses a variety of different techniques to prepare DNA fibers from all the genetic material of the cells to be studied, that is, DNA analyze.
The key to fiber-FISH is the preparation of high-quality linear DNA fibers. Ideally, the length of the prepared DNA should be similar to that of a fully stretched DNA fiber, and there should be as few breakpoints as possible. Various methods for preparing DNA fibers have been developed in recent years. Fiber-FISH can carry out quantitative analysis, requires a small amount of template and does not require high requirements, and has the advantages of high resolution and high sensitivity. Therefore, fiber-FISH plays a very important role in chromosome mapping, gene recombination research and clinical chromosomal gene sequence detection.
As a molecular cytogenetic technique to visualize specific DNA sequences, fluorescence in situ hybridization is currently widely used for chromosomal aberrations. Such as aneuploidy, chromosomal recombination. The basic process includes probe labeling, probe denaturation, sample denaturation, hybridization and fluorescence signal collection.
Fluorescence in situ hybridization technology has advantages in the research of gene characterization, quantification, integration and expression. It has been widely used in many fields such as genetic disease diagnosis, viral infection analysis, prenatal diagnosis, tumor genetics and genome research. It plays an important role in clinical testing, teaching and research.
(1) Gene (or DNA fragment) chromosomal location and gene mapping
The main method of gene mapping currently used is FISH. The isolated DNA sequences can be directly passed through FISH, and the labeled probes of multiple colors of fluorescein can be used at the same time. Combined with the information of metaphase chromosomes and interphase cells, the mutual order and distance between a series of DNA sequences can be quickly determined, and the gene can be completed. mapping. When two different DNA strands are labeled with different color ray 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), high-resolution banded chromosomes can be obtained, and the sorting ability of DNA strand markers to chromosomes can be improved. If using interphase cells, the distance between the two DNA strands can be shortened to 50kb, which is 1/20 of the resolution distance on the chromosome, and the order of different probes can be determined by measuring the distance in interphase cells. Determining the precise location of DNA strands on chromosomes is suitable for detecting translocations and deletions in some special chromosomal conditions. By labeling the same DNA strand and the chromosomes of cells of different species, it is possible to find out the homologous genes between different species and the position of genes on the chromosome, so as to understand the evolutionary relationship between species.
(2) Abnormal number and structure of chromosomes
In cytogenetic examination, the probes of repetitive sequences are the most widely used, including α-satellite DNA probes, β-satellite DNA probes and classical satellite DNA (elassic-stllite DNA) probes. a Satellite DNA probe mainly detects the centromere of human chromosomes. β-satellite DNA probes are located around apical centromeric chromosomes and the heterochromatin of chromosomes. Classical satellite DNA probes have short repeats of AATCG located around chromosomes 1, 9, 15, 16 and heterochromatin on the long arm of the Y chromosome. The latter two probes can be used not only for the detection of chromosome number, but also for the detection of fine changes in the above-mentioned parts. The application of FISH technology to detect chromosome number and structural abnormalities has high specificity and sensitivity, and has been widely used in rapid prenatal diagnosis.
(3) Hematology and Oncology
The clinical FISH detection of hematological tumors mainly focuses on: the detection of fusion genes formed by chromosomal translocation, such as ber/abl translocation DNA probe, t(15;17) translocation DNA probe and t(18;21) Translocation DNA probes, etc.; gene deletion detection can find the deletion of some key genes, which is helpful for the diagnosis and prognosis of the disease; the use of fluorescence in situ hybridization technology can detect minimal residual lesions and monitor the status of hematopoietic stem cell transplantation .
(4) Solid Oncology
All the methods for measuring gene amplification before FISH technology used classical molecular biology methods. Compared with FISH, these methods are not only time-consuming and labor-intensive, but also cannot observe the state of gene amplification at the cellular level. The greater advantage of FISH technology is that direct evidence of DNA amplification can be observed in interphase nuclei, and the quantity and fluorescence intensity of amplified DNA fluorescent signals 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 lymphoma. Amplification of the Her-2/neu gene in breast cancer cells often predicts poor patient prognosis, and 25% to 30% of breast cancer patients have Her-2/neu gene amplification and/or overexpression. The use of Her-2/neu gene DNA probe to detect the amplified expression level of Her-2/neu gene is beneficial to the clinical diagnosis and curative effect monitoring of breast cancer. The use of chromosomal centromere-specific probes can be used to analyze the number of chromosomes in interphase cells. For example, Hopman used FISH technology to study bladder cancer and found the loss of chromosome 9. Using a variety of chromosomal probes, marked with different colors, can be used to study the heterogeneity of tumor chromosome number changes. At present, FISH is mainly used for early diagnosis, curative effect detection, individualized treatment and prognosis judgment of tumors.