Microarray - comparative genomic hybridization technique for detecting chromosomal abnormalities
ã€Abstract】 In recent years, microarray-comparative genomic hybridization (microarray. CGH) technology has been applied to the field of clinical cytogenetics. The technique is to select a specific fragment of DNA as a target and solidify on a carrier to form a dense, ordered molecular microarray. Then, DNA is extracted from the test specimen, and the test DNA and the reference DNA are labeled with different fluorescent pigments, hybridized to the microarray, and the change in the copy number of the specimen to be tested is known by detecting the ratio of the two fluorescent pigments. It is capable of detecting submicrostructural abnormalities of chromosomes. Microarray technology has become an important tool for the diagnosis of chromosome submicrostructural abnormalities before, during and after prenatal implantation.
In the past 30 years, karyotyping has been the standard method for diagnosing chromosomal disorders. In recent years, microarray-comparative genomic hybridization (microarray-CGH) technology has been used in chromosomal disease research. This technique is also known as "molecular karyotyping." Can diagnose chromosome submicroscopic abnormalities. This article focuses on the application of microarray-CGH technology in cytogenetics. microarray-CGH technology introduction
First, the technical principle and development
microarray-CGH technology is a molecular cytology technology developed in recent years. Although the traditional comparative genomic hybridization (CGH) technology is based on the same principle, the traditional CGH is a medium-term split phase. And microarray. CGH is a specific fragment of DNA selected as a target, which is solidified on a carrier to form a dense, ordered molecular microarray. Because a particular fragment of DNA is targeted, the resolution can be determined based on the size and density of the DNA fragment. microarray-CGH detects micro-repeats and deletions that cannot be detected by traditional methods. This whole genome array detection is referred to as "molecular karyotyping" in comparison to traditional karyotyping. initial. microarray-CGH was introduced into the field of tumor cytogenetics research because of the difficulty in culturing tumor cells and the poor form of metaphase division. And tumor cells have complex chromosome changes,
Traditional chromosome analysis methods are difficult to detect small deletions and duplications of genetic material. The use of whole-genome microarray-CGH to study a large number of tumor specimens can map areas where gene copy number changes frequently occur. A high-density microarray-CGH can be used to analyze the gene copy number changes in these regions. Tumors can be typed according to the type and number of changes in gene copy number to identify genes that cause tumorigenesis. And recently the technology has been used for
Bed cytogenetics, detection of clinically expressed chromosomal microdeletions and microrepetition syndrome Es-9].
Second, technical characteristics
Molecular karyotypes show many advantages over traditional karyotypes. However, microarray-CGH is not a substitute for traditional chromosome analysis because it cannot detect balanced translocations and polyploids of chromosomes. microarray-CGH was found to be abnormal by detecting changes in gene copy number in patients and controls. There is no change in the copy number of the gene in the balanced translocation, and therefore, the balanced translocation cannot be detected. In addition, microarray-CGH identifies abnormalities by detecting the ratio of fluorescence intensities, and the fluorescence intensity of the double dye from triploid is finally considered normal, so polyploids cannot be detected.
In recent years. microarray-CGH has been used in clinical cytogenetics research, and the results are reviewed in three aspects. Detection of aborted villus chromosomes Currently, clinical use of chorionic tissue to prepare chromosomes for diagnosis of abortion embryos has chromosomal abnormalities. There are usually some problems with the culture of villus cells. Including: high-risk culture failure, excessive growth of mother cells, poor chromosome morphology, etc. Therefore, although the fetal chromosome is abnormal. Sometimes it is hard to find. Schaeffer et al. first published the use of microarray-CGH technology to examine the chromosomes of aborted embryos in 2004. In this study, the DNA of choice. Array includes terminal telomeres for each chromosome, all special fragments of DNA for minor deletion syndrome. Also choose some special
Special site DNA fragment. Analysis of 41 aborted embryos, microarray-CGH not only detected all abnormalities found in chromosome G-band analysis, but also found 4 cases of chromosome submicroscopic abnormalities. Subsequently, Shimokawa et al. also published a similar report. In 20 routine chromosomal analyses showing normal karyotype aborted villus samples, microarray-CGH analysis revealed that one of them had short arm microdeletions of chromosome 3. Studies by Benkhalifa et al. and Fritz et al. showed that microarray-CGH has the ability to detect chromosomal abnormalities in non-proliferating cells in culture. therefore. It detects chromosomal abnormalities that cannot be found by conventional chromosome analysis.
microarray-CGH can not only detect chromosome submicrostructural abnormalities, but also detect it by using the DNA extracted from the specimen. microarray-CGH also solves the problems caused by the failure of villous cell culture. If it will be microarray. The application of CGH to the examination of chromosomes in aborted embryos will result in faster and more accurate diagnosis. Detection of multiple malformations. Chromosome abnormalities in mentally retarded children In recent years, microarray-CGH technology has also been applied to multiple malformations in children with low intelligence. LeCaignec et al. used a commercial array kit (including the end of all chromosomes, the major chromosomal microdeletion syndrome and 201 areas covering the whole genome) to detect 49 children with more than 3 malformations but normal karyotypes. 8 submicrostructural anomalies, including terminal and internal deletions. Submicrostructural duplication and complex genetic imbalances. There was a clear association between the 4 genotypes and the phenotype. The 6q terminal microdeletion was detected in 1 mother and 2 of her multiple malformed infants, which made the relationship between genotype and phenotype unclear. In addition, 3 cases monitored micro-repetition of 10q. This may be a genetic polymorphism. The results show that microarray-CGH can detect the submicroscopic structural abnormalities of chromosomes. The genetic polymorphism of some genes can also be found.
Schoumans et al [] use microarray. CGH detects a group of children with unexplained mental retardation. These 41 patients had unexplained mental retardation and multiple malformations, and no abnormalities were found on routine chromosome examinations. Thirty cases of multi-terminal site in situ fluorescence hybridization (FFU) were screened, and 11 cases were analyzed for spectral karyotyping (Sky). The 41 samples were tested using a commercial kit (2600 BAC clones scattered over the entire genome at 1 Mb intervals). Four cases were found to have chromosome submicroscopic structural abnormalities, and the length of the abnormal gene fragments ranged from 1 to 14 Mb. Nowakowska et al [] further confirmed microarray-CGH
The role of technology in monitoring chromosome submicrostructural abnormalities. Using chromosomal microarray analysis (CMA) technique to analyze 9 cases of children with low intelligence but normal karyotype, 19 (20.8%) children had gene copy number changes, of which 11 cases (11.8) %) Gene copy number changes were associated with clinical manifestations, and 6 (6.5%) were considered to be genetic polymorphisms. The change of gene copy number in 2 cases (2.1%) was unknown, and the length of the abnormal gene fragment was between 0.5 and 12.9 Mb. Recently, Tsuchiya et al. used microarray-CGH technology.
Four patients with extra-marker small chromosomes were found in the study. The structure of the extra small chromosome is much more complicated than the expected structure, and other molecular cytogenetic methods cannot be analyzed in detail. Microarray. The application of CGH technology in the field of cytogenetics will help to analyze the extra small chromosomes and further understand its relationship with phenotype.
Application in prenatal diagnosis
Chromosome karyotyping has been used in prenatal diagnosis for more than 30 years and has been considered a standard prenatal diagnostic method. Although karyotyping can accurately identify aneuploidy and large chromosome structural abnormalities, this technique has significant limitations in resolution. Another disadvantage is that there are few viable cells in the amniotic fluid, and cell culture must be performed for 1 to 2 weeks before karyotyping. The patient waits for a long time to wait. High-risk pregnancies require a quick test. And microarray. CGH technology can overcome these shortcomings. If microarray-CGH can be used for uncultured cells. Can greatly shorten the time patients wait for results. To this end, Larrabee et al. performed microarray-CGH analysis using free DNA extracted from amniotic fluid. In the study, the free DNA of 11 male fetal amniotic fluids was first investigated. Compared with the female fetal DNA, there was a significantly enhanced positive signal and a reduced x chromosome specific site signal in the SRY gene. Then, 3 cases of 21-trisomy fetuses were investigated. Compared with diploids, the signal at the special site of 21-trisomy was significantly increased. In addition, 1 case of x-unit fetus was investigated, and special sites on most x chromosomes were investigated. The signal is significantly reduced. The results showed that the free DNA was extracted from the amniotic fluid and analyzed by microarray-CGH to correctly diagnose the abnormality of the sex and chromosome number of the fetus. And this technology will likely become a method of screening the entire fetal genome.
microarray-CGH can also detect genetic polymorphism while monitoring chromosome submicrostructural abnormalities. Therefore, its resolution and composition should be carefully considered when applied to prenatal diagnosis. If you use a very high resolution array. Some changes in gene copy number that are not related to clinical manifestations may be detected. A low-density array was chosen to cover the entire genome with an average resolution of 10 Mb, but increased density in some areas to detect minor deletions and minor repetitive syndromes and subterminal deletions. Rickman] investigated 30 prenatal and postnatal specimens in this way, except for one triploid, which was detected.
All abnormalities that have been detected by karyotyping and FISH. In addition, the detection rate was improved as compared with the 1 Mb array. recent. Shaffer et al [plus] with microarray. CGH technology detected chromosomal abnormalities in 1.3% of 151 prenatal specimens: 11.4% of 1375 neonates had chromosomal abnormalities. The difference in the detection ratio is considered to be due to microaray-CGH detection in 40% of newborns due to abnormalities. Ultrasound and prenatal screening cannot detect these abnormalities. If microaray-CGH is applied to prenatal screening, then. Prenatal diagnosis
More abnormal fetuses will be found.
The characteristics of microaray-CGH technology make some chromosome submicroscopic abnormalities diagnosed in the uterus. At the same time, the detection of DNA does not require cell culture, and the diagnosis results can be quickly obtained. Therefore, this technology has broad application prospects in the field of future prenatal diagnosis.
Prospects
Microarray. The CGH technology makes it possible to detect submicrostructural abnormalities of chromosomes. Moreover, it is possible to achieve rapid and automatic detection of specimens. It will play an important role in the field of cytogenetics. Diseases caused by abnormalities in chromosome submicrostructures that cannot be detected under current conditions are diagnosed. At the same time, the time and labor required for inspection can be greatly reduced. To a large extent will replace the karyotype analysis. Since the balanced translocation and polyploidy of chromosomes cannot be detected, Traditional chromosomal examinations will also remain in the diagnosis of chromosomal disorders.
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