Chromosomal in situ fluorescent molecular hybridization technology provides a most direct method for studying the sequence of DNA on chromosomes.
The development of Chromsome Analysis by Fluorescencein situ Hybridization in tissue sections provides a most direct method for studying the sequence of DNA on chromosomes. It has the advantages of economy, safety, rapidity, stability and high sensitivity. Colorful FISH can display two or more sequences in the same nucleus, and can also study interphase nuclear chromosomes (Figure 1). All genes of a species, stained fragments of a chromosome and single-copy sequences; combined with confocal laser microscopy, the three-dimensional structure of interphase nuclei and chromosomes can be studied, and hybridization signals can be accurately detected.
1. Genomic probe:
Some high-frequency repetitive sequences in the human genome are rarely conserved in evolution. If genomic DNA is used as a probe, these highly repetitive sequences present in the probe and the target cell sequence will first anneal and bind, bypassing those conserved and unique sequences, so the hybridization will show species specificity. Human genetic material can be specifically displayed in human-mouse hybrid cells using such probes.
2. Chromosome-specific sequence probes (proberecognizingchromosomespecificsequences):
Some repeat sequences have been cloned in almost all human chromosomes, ranging from 100 to 5,000 times, each with chromosome specificity. Most of them produce dense hybrid bands in the centromeric region or heterochromatin region of a chromosome. Thus, a chromosome can be specifically identified.
3. Chromosome library probe (chromosomelabrariesprobe):
The chromosome library collects the DNA of a single human chromosome as a probe, also known as the whole chromosome probe. There are generally two ways to obtain a single chromosome: from a somatic hybrid strain carrying only one human chromosome, or from a chromosome suspension by flow cytometry.
4. Single-copy sequence probes:
FISH has a weakness, that is, the probe is required to be large enough. The smaller the probe, the lower the detection rate of hybridization sites. To use FISH to detect a single sequence of genes present in the genome, the most efficient method is to use cloning vectors containing large inserts, such as whole Cosmids (carrying about 40kb inserts), larger YAC vectors (carrying 100-800kb inserts) ). If mastered well, plasmid probes as small as 2kb can also be located by FISH, but the efficiency is low.
FISH technology series
1. 24-color mFISH karyotyping technology
24-color mFISH is a new technology. The principle is to use 5 kinds of fluorescent dyes to label the probes in proportion, and after hybridization, 24 chromosomes are formed, each showing a specific fluorescent color for karyotype analysis. It provides researchers with richer and more detailed cytogenetic information, including determining the source of marker chromosomes, detecting small chromosomal translocations and detecting complex chromosomal translocations, especially providing a new and efficient method for chromosomal analysis of tumor cells.
24-color chromosome mFISH karyotype analysis of esophageal cancer cell lines
2. In situ hybridization banding technology (insituhybridizationbanding, ISHB)
In order to accurately locate the chromosome and its zone where the in situ hybridization site is located, the chromosome must be banded. However, whether it is first hybridization and then banding, or first banding and then hybridization, the process of hybridization and banding will affect each other. It was later noticed that an Alu family of human short-spaced insertion repeats, the Alu fragment is about 300bp long, repeated about 900,000 times in the genome, and one is inserted at an average interval of 3-4kb. Some people use part of the Alu sequence as a primer to amplify the DNA between Alu by PCR method, which is called Alu-PCR method. However, the distribution of Alu sequences in the genome is not random, some regions are dense, and some regions are sparse. Only the former had PCR products. People use the Alu-PCR product as a probe to hybridize with human chromosome specimens, and the result is a fluorescent band pattern similar to the R band. Therefore, when people conduct gene mapping, they only need to apply the target probe and the Alu-PCR probe at the same time, and use fluorescent labels of different colors to display the hybridization signal and the chromosome band type at the same time.
3. FISH gene mapping (FISHmapping)
In gene mapping, it is not only necessary to determine the position of a certain target sequence on the chromosome, but also to determine the order and distance of two or more target sequences in the linear DNA molecule, in order to draw a gene map. Isotope hybridization is generally used: first determine the position of each target sequence on the metaphase chromosome, and then determine the linear order according to their distance from the telomere. With FISH, two or more probes can hybridize to metaphase chromosomes at the same time, and the order can be determined directly based on the mutual positions of the hybridization sites of the two colors. But metaphase chromosomes are formed by the folding and packaging of linear DNA molecules. If the two target sequences are very close to each other, for example, the distance is less than 1Mbp, their arrangement on the linear DNA molecules is different from that on the metaphase chromosomes due to the influence of the packaging process. Not necessarily the same, maybe even the exact opposite. The scholars found that the average relative distance between target sequences in the interphase nucleus was positively correlated with their distance on the linear DNA molecule. Using interphase nuclear FISH analysis can not only exclude the influence of chromosome packaging, but also improve the resolution of ranging.
Single chromosome coating of esophageal cancer cells, and site-specific mapping by BACFISH
4. Application of FISH
(1) The principles of gene mapping and genemapping have been described above. FISH has greatly accelerated the progress of human gene mapping and gene mapping.
(2) Accurate, intuitive and clear genetic diagnosis
(3) Interphase cytogenetics
(4) Application of FISH in tumor biology
① Tumor cytogenetics (onco-cytogenetics);
② Gene localization: FISH can be used for preliminary localization of isolated oncogenes and tumor suppressor genes;
③ Detection of viral gene insertion into the genome: Although retroviruses mainly activate proto-oncogenes, there are also viruses such as adenovirus, HPV, and SV40 that interact with tumor suppressor gene protein products to induce cell transformation. However, viral genes, especially DNA viruses, often have viral gene components inserted into the human genome. The integration of the virus into the human genome can be detected by FISH, which is of great significance for the in-depth study of the carcinogenic mechanism of the virus, as well as the detection and prevention of tumors;
④ Gene amplification and deletion: The activation of proto-oncogenes and the inactivation of tumor suppressor genes are hotspots in tumor research. The known activation methods of proto-oncogenes include: mutation, gene amplification, translocation, and insertion of viral sequences. The inactivation methods of tumor suppressor genes include point mutation and gene deletion. FISH provides a new method for studying gene amplification and deletion, which can separate gene amplification and chromosomal duplication.