Which molecular method uses fluorescent probes to evaluate genes and or DNA sequences on chromosomes?
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The study of the genetics of cells and molecular biology has helped us to build up some ‘in situ hybridization’ techniques [1], through which congenital disorders or abnormalities can be detected to handle the cases more effectively and efficiently during the clinical practices [2]. The level of accuracy and specificity in these diagnoses play the pivotal role in treating, curing, preventing, and lessening the pain and agony of the patients as well as pave the way for further clinical developments in these fields of medical sciences [3]. Show The detection of specific gene sequences on the chromosome, or either its presence or absence, is the central concern of cytogenetic technique in diagnosing as well as enumerating a genetic disorder or abnormalities. Among the tools of cytogenetic techniques, a technique named fluorescence in situ hybridization (FISH) was developed in the early 1980's [4]. Applications of the FISH assay have been on the rise since the 1990's. Fluorescent deoxyribonucleic acid (DNA) probes, which are attached to the high degree of complementary parts of the chromosome, emit the colored signals. These signals could be grasped and visualized using fluorescent DNA probes, which further unveiled another way of detecting genetic abnormalities in medical science [5]. FISH is a less time-consuming technique in comparison to the conventional method of cytogenetic metaphase karyotype analysis [6] because, in the former, the detection technique involves processing with either fresh water or paraffin-embedded interphase nuclei without the need of culturing [7]. By using this technique, specific cytogenetic abnormalities, as well as a copy of aberrations numbers, can be enumerated and sketched. For example, chromosomal microdeletion, amplification, and subsequently, translocation can easily be detected through FISH. The improved diagnostic techniques have made the treatment easier and increased the life expectancy of people [8]. Therefore, FISH is becoming a more vital tool to detect and monitor the specific therapy with regards to the gene abnormalities; for example, the detection of the BCR/ALB1 translocation in chronic myeloid leukemia, human epidermal growth factor receptor 2 (HER2) augmentation in breast cancer, and anaplastic lymphoma kinase (ALK) rearrangement in adenocarcinoma [9-10]. The aim of this article is to review the concept of FISH, its growing need for application, and its advantages in medical science. Mechanism of FISHThe Basic Elements of FISH: DNA Probe and a Target Sequence The first step is to prepare short sequences of single-stranded DNA that match a portion of the gene that we are looking for. These are called probes. The DNA probe is labeled in various ways, such as nick translation, random primed labeling, and PCR. Two labeling strategies are used: indirect labeling and direct labeling. In the case of direct labeling, probes are being labeled with nucleotides containing a fluorophore. In indirect labeling, it is the modified nucleotides that contain a hapten at which probes are being labeled. Then, the labeled probe and the target DNA are denatured. The annealing of complementary DNA sequences happens due to the combining consequences of denatured probe and target DNA. In the case of indirect labeling, an extra step is needed for visualization of the non-fluorescent hapten that uses an enzymatic or immunological detection system [2]. However, the selections of the FISH probe are dependent upon the diseases, anomalies, or anomalies under the field of interest [11]. Basic steps of the FISH procedure is depicted in Figure 1.  MethodologySearch Strategy and Keywords A literature search was done in PubMed, PubMed Central, and Google Scholar using strings of keywords as follows: fluorescence in situ hybridization, genetic anomalies and their correction, and application of FISH with dates ranging from January 1984 to December 2015. After completion exclusion of repetition was done, then articles were chosen purposively according to the inclusion and exclusion criteria. From total 3,024 articles, 57 articles were finally selected for review. Inclusion and Exclusion Criteria We included the articles from 1984 to 2015, which had full text in PDF format and were available in English. Articles without quantitative data and whose outcome evaluation methods were different were also excluded. Data Extraction The total of 3,024 articles was found. After exclusion of repeated articles (1,346), 1,678 articles were left. From the 1,678 articles, 854 article were selected by screening the title and abstracts manually. Eight hundred and fifty-four articles were screened and 217 were selected for review after assessing the full text. Finally, 57 articles were selected after applying the exclusion criteria (Figure 2). Reliability Assessment Reliability was assessed in forms of the internal consistency, test-retest reliability and inter-rater reliability, and split-half reliability. Most of the articles assessed the internal consistency form of reliability, and in all articles, reliability was measured by Cronbach’s alpha having a level of ≥ 0.70. ResultsDifferent types of FISH and their function are shown in Table 1.
Increasing trends of FISH technology around the worldWe carried out a search for citations in the PubMed database using the keywords ‘fluorescence in situ hybridization’, 'genetic anomalies and its correction', and "application of FISH" by publication year. We found that the technique, fluorescence in situ hybridization (FISH), was first introduced in the early 1980's. The FISH assay gained popularity after the 1990s. A new arena of research was started by this application (Figure 3). Review of the FISH technology in medical scienceHistiocytoid Sweet Syndrome Histiocytoid Sweet syndrome which is known as acute febrile neutrophilic dermatosis was initially described by Robert Douglas Sweet. It is a dermatological disease with an underlying neutrophilic infiltrate. Sweet syndrome is closely associated with an either or combination of the underlying hematologic myeloid disorder or solid tumor malignancies or inflammatory bowel disease or gastrointestinal tract or upper respiratory tract infections. Here, FISH was performed to determine the presence of BCR/ABL gene fusion. Traditional approaches cannot fulfill the diagnostic criteria of the sweet syndrome. Hence, the FISH was conducted to assess the presence of a chromosomal abnormality in the cutaneous infiltrate of the initial biopsy specimen. Histiocytic Sweet syndrome may indicate leukemia cutis as well [37]. Differential Diagnosis of Pseudomosaicism from True Mosaicism Interphase FISH in uncultured amniocytes provided a rapid differential diagnosis of pseudomosaicism from true mosaicism. However, it is fetal abnormalities to which true mosaicism for isochromosome 20q has been associated. On uncultured amniocytes, molecular cytogenetic analyses by interphase FISH can be used as a vital tool for a differential diagnosis of pseudomosaicism from true mosaicism in isochromosome 20q detection [38]. Autologous Fat Grafting Autologous fat grafting has been widely used in many reconstructive surgeries, such as aesthetic skeletal remodeling, breast reconstruction, facial rejuvenation, and facial reconstruction [39]. FISH can be used to detect specific DNA or ribonucleic acid (RNA) sequences within the context of the cell. Here, the technique may help the surgeon to determine the degree of angiogenesis and adipogenesis that arises from the recipient versus grafted cells. Dedifferentiated Liposarcoma (DDLPS) For a better prognosis in distinguishing dedifferentiated liposarcoma (DDLPS), it is important to distinguish DDLPS earlier from other high-grade spindle and pleomorphic sarcomas. Mouse double minute 2 homolog (MDM2) amplification and expression are potentially crucial in distinguishing between DDLPS and other undifferentiated high-grade spindle and pleomorphic sarcomas. MDM2 fluorescence in situ hybridization provided excellent data for distinguishing the diseases [40]. Streptococcus Pneumonia Streptococcus pneumonia can be detected through FISH from blood culture samples. Streptococcus pneumonia (pneumococcus) plays as a major causative agent for bacteremia in both children and adults [41]. However, the FISH procedure without enzymatic treatment can identify S. pneumonia in blood culture specimens. Studies in Bangladesh found that the new technology could be a useful intervention to detect newborn specific disease [42]. Aneuploidies Studies strongly suggested that the age-related aneuploidies are mainly due to nondisjunction occurring during maternal meiosis [43]. However, the study of first and second polar bodies with the help of one of the cytogenic methods, enable us to detect aneuploidy oocytes in IVF patients. Thus, it can help us to formulate a way to prevent the transfer of embryos resulting from aneuploidy oocytes. It may reduce the chances of an IVF couple to have a child with Down syndrome and other common aneuploidies. With the help of FISH, the detection of chromosome signals in interphase nuclei is possible. Hence, this may be a reliable method for detection of common aneuploidies before the implantation takes place [44]. Application of FISH in oncologyChronic Myeloid Leukemia (CML) The abnormal fusion proteins resulting from chromosomal rearrangements have remarkably contributed in the molecular mechanism of leukemia. The BCR/ABL1 translocation often occurs in chronic myeloid leukemia (CML). The FISH assay is used as the gold standard for detecting these chromosomal translocations, and thus, it can be used as a vital tool in selecting a targeted therapy in different leukemias [45]. Multiple Myelomas (MM) Multiple myelomas are the heterogeneous malignancy of terminally differentiated B cells. Molecular studies suggested that primary translocations occur in the early stage of MM, followed by a huge number of secondary translocations in the time of tumor progression [46]. Here, FISH is effective for analysis of interphase nuclei and small chromosomal aberrations, which are recognized as the most vigorous genetic test for characterization of cytogenetic abnormalities in MM. Pulmonary Adenocarcinomas Anaplastic lymphoma kinase (ALK) rearrangements are related with pulmonary adenocarcinomas. ALK rearrangements are mostly found from the fusion of the echinoderm microtubule-associated protein like 4 (EML4) with ALK at chromosome 2p23 [47]. EML4-ALK gene fusion can be detected through FISH. Prostate Cancer In half of the prostate cancer cases, androgen-regulated TMPRSS2 and E26 transformation-specific (ETS) family members (ERG, RTV1, and ETV4) were detected [48]. Invariably, chromosome rearrangement involves the fusion of TMPRSS2 to the oncogene ETS-related gene (ERG), which leads to the abnormal activation of ERG. In recent years, a four-color FISH assay was used for the detection of either TMPRSS2 or ERG rearrangements. Breast Carcinomas In 10% to 20% of human breast carcinomas, overexpression of HER2 is observed [49]. The HER2 protein is an active tyrosine kinase which plays a major role in normal cell growth and differentiation. The HER2 status is crucial for the recommended targeted therapy. At present, FISH assays are used for measuring HER2 overexpression. The FISH assay is regarded as a vital tool for clinical evaluation of the HER2 status. Renal Mesenchymal Neoplasm Appropriate identification of renal mesenchymal neoplasms is challenging. Diagnosis can be difficult with needle biopsy; here, immunohistochemistry [38] and in-situ hybridization are used for the accurate diagnosis. Cholangiocarcinoma (CC) Cholangiocarcinoma (CC) is regarded as a rare malignancy of the gastrointestinal tract with a poor outcome. Because of the limited entrance to the tumor site, the preoperative diagnosis depends upon the interventional imaging techniques, mainly, the endoscopic retrograde cholangiopancreatography (ERCP) [50]. However, it is also difficult to collect suitable tissue samples. Thus, obtaining genetic information of CC is hampered. Studies showed that the alternative FISH technique on brushing smears can detect numerical and structural abnormalities of four chromosomes in patients with documented extrahepatic CC. Melanoma Melanoma is a type of cancer that develops from the pigment-containing cells known as melanocytes. It can be accurately detected through FISH. For the diagnosis, four probes targeting 6p25 (RREB1), 6q23 (MYB), 11q13 (CCND1), and centromere 6 (CEP6) are used. The optimal algorithms for detecting positive FISH results based on these four probes are also established [49]. Limitation of the reviewThe present article systematically reviewed the present literature to show the benefits of using FISH. However, meta-analyses should be conducted in future to prove the benefits of FISH over the other conventional techniques. In comparing a regular application of clinical diagnosis, the FISH is much more accurate and straightforward, as well as reliable, than the other molecular profiling techniques, e.g., array-based comparative genomic hybridization, single nucleotide polymorphism (SNP), etc. Moreover, FISH gained great recognition as a physical mapping technique to support large-scale mapping and sequencing efforts related to the human genome project. We can visualize and conceptualize genes, chromosomes, transcription, and nucleic acid movements through FISH. FISH also provides reliable biomarker information. In terms of using the FISH technology around the world, we found that the FISH technique has very limited use in developing countries because of unavailability and lack of expert knowledge. Hence, FISH should be the preferred approach in anticipating the complex components of gene expression leading to any disease. What is fluorescent DNA analysis?Fluorescence in situ hybridization (FISH) provides researchers with a way to visualize and map the genetic material in an individual's cells, including specific genes or portions of genes. This may be used for understanding a variety of chromosomal abnormalities and other genetic mutations.
What is the fluorescence in situ hybridization test?Fluorescence in situ hybridization (FISH) is a cytogenetic technique developed in the early 1980s. FISH uses fluorescent DNA probes to target specific chromosomal locations within the nucleus, resulting in colored signals that can be detected using a fluorescent microscope.
What is FISH technique used for?Fluorescence in situ hybridization (FISH) is the most convincing technique for locating the specific DNA sequences, diagnosis of genetic diseases, gene mapping, and identification of novel oncogenes or genetic aberrations contributing to various types of cancers.
How is fluorescent in situ hybridization done?General process of fluorescent in situ hybridization (FISH) used for bacterial pathogen identification. First, an infected tissue sample is taken from the patient. Then an oligonucleotide complementary to the suspected pathogen's genetic code is synthesized and chemically tagged with a fluorescent probe.
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