Application of FISH

Fluorescence in situ hybridization (FISH) was introduced almost 30 years ago, it was a beginning of a new era in the study of chromosome structure and function. FISH is a very straightforward technique. FISH is a technique in which DNA probes are hybridized to its complementary sequence on the chromosome which is prepared and fixed on a slide. The chromosomes held on slides are pre-treated suitably so as to expose and denature their DNAs without actually affecting their structural integrity. The probes used can be directly labelled by incorporation of florescent nucleotides or can be indirectly incorporated in the reporter molecules which are subsequently detected by fluorescent antibodies or other affinity molecules. FISH is a combined molecular and cytological approach. It has a major advantage of providing intermediate degree of resolution between DNA analysis and chromosomal investigation, while also retaining information at the single- cell level. The technique is accurate and adaptable, so it can be exploited in various biological and medical researches as well as in diagnostic assay using simple modifications.

FISH finds applications in various fields as follows:
1) In medical sciences

• Histiocytoid sweet syndrome.
Histiocytoid sweet syndrome is also called acute febrile neutrophilic dermatosis is a dermatological disease with an underlying neutrophilic infiltrate. The traditional methods do not fulfil all the diagnostic criteria. So FISH is used in to determine the presence of BCR/ABL gene fusion. Any abnormalities in the chromosome of the cutaneous infiltrate of the initial biopsy specimen can indicate the presence of the syndrome.

• Autologous fat grafting
Autologous fat grafting is used in reconstructive surgeries such as breast reconstruction, facial rejuvenation, facial reconstruction, etc. FISH helps in detection of DNA or RNA sequences within the context of cells. This helps the surgeon to determine the degree of angiogenesis and adipogenesis that arises from the recipient versus grafted cells.

• Presence of Streptococcus pneumonia in blood sample: By using FISH we can detect the presence of Streptococcus pneumonia in blood samples. S. pneumonia is the causative agent of bacteremia in both children and adults. The procedure of FISH can identify S. pneumonia in blood samples without enzymatic treatment.

• Aneuploidies
The main reason for age- related aneuploidies is due to nondisjunction occurring during maternal meiosis. Cytogenic methods enables us to detect aneuploidy oocytes in IVF patients and prevent the transfer of embryos which have resulted from aneuploidy oocytes. This decreases the chance of IVF couple to have a child with Down’s syndrome and other common aneuploidies. The detection of chromosome signals in interphase nuclei is possible using FISH. FISH is a reliable method to aneuploidies before the implantation takes place.

• Prader – Willi syndrome and Angelman syndrome.
The Prader – Willi syndrome and Angelman syndrome are different disorders, which are caused by abnormalities in the chromosome 15q11-q13 regions. Prader- Willi syndrome patients show cytogenetic or molecular deletion of the paternally inherited chromosome 15. Most of the non-deletion cases show maternal uniparental disomy. Micro deletion of the maternal chromosome 15 is seen in patients with Angelman syndrome. FISH has been used for the conformation of the presence or absence of the chromosome. FISH also has an additional advantage as it can be used on interphase nuclei allowing even faster diagnostic conclusions

Plasmodium spp.
Few segments of the 16S rRNA are invariant in all organisms but phylogenetic group specific 16S rRNA in different groups of organism can be used as oligonucleotide FISH probes. These probes can be used to identify infectious agents having the specific sequence in the clinical sample. The specific sequence of the 16S rRNA present in Plasmodium spp. can be used to detect the presence of the organisms in the blood sample by using FISH probe which is complementary to that specific sequence. Such probes are available for all the five species of Plasmodium known. As rRNA has a short life and is present in plenty copies in living organisms, FISH can be used to detect the live pathogen.

2) In oncology
• Chronic Myeloid Leukemia (CML)
Chromosomal rearrangement has caused abnormal fusion proteins which contributes in the molecular mechanism of leukemia. The translocation of BCR/ABL1 gene leads to CML. FISH is used as a gold standard for detection of chromosomal translocations. FISH is a vital tool in selecting a target- based therapy in different leukemias.

• Multiple myelomas (MM)
Multiple myelomas is a condition of heterogeneous malignancy of terminally differentiated B cells. According to various molecular studies the primary translocation of occurs in the early stages of MM, which is then followed by a huge number of secondary translocations during the progression of the tumor. FISH technique is very effective for the analysis of interphase nuclei and small chromosomal aberrations, this is considered as the most vigorous test to characterize cytogenic abnormalities in multiple myeloma.

• Breast carcinomas
In most of the cases of breast cancer, there is an overexpression of HER2 observed. The major role of normal cell growth and differentiation is regulated by HER2 which is an active tyrosine kinase. This overexpression of HER2 can be measured using FISH assay.

• Melanoma
The cancer which develops in the pigment containing cells i.e. melanocytes is known as melanoma. 6p25 ( RREB1), 6q23 (MYB), 11q13 (CCND1) and centromere 6 (CEP6) are the probes used for diagnosis using FISH. Melanoma can be accurately detected using FISH.

3) In prenatal diagnosis

• To identify low levels of mosaicism in Ullrich- Turner syndrome patients.
Ullrich Turner syndrome is identified by abnormalities in the chromosomes in human. In humans it is characterized by short stature, streak gonads, primary amenorrhea, somatic abnormalities, etc. The identification of low-level Y chromosome mosaicism is clinically very important as, the patients with Y chromosomes have about 15-20% risk of developing gonad blastoma. FISH is a direct approach which helps us excess and identify even low levels of sex chromosome mosaicism in many patients. This helps in better counseling and clinical management of the patient.

• Pre-implantation genetic diagnosis (PGD)
Pre -implantation genetic diagnosis involves selecting pre-implantation embryos from a cohort generated by assisted reproduction technology. These selections are done because of familial monogenic disease or because one of the partners carries a chromosomal rearrangement. PGD is available for couples that have previously affected children or in case of recurrent miscarriage or infertility. Oocytes aspirates are taken after ovarian stimulation. Pre-implantation cleavage stage embryos are biopsied, normally by removal of a single cell on the day 3 post-fertilization and the genetic status of the embryo can be studied using FISH. FISH of the biopsied cells with target specific DNA probe is used to detect the chromosomal imbalance associated with X- linked diseases.

• Chromosomal analysis of spermatozoa.
Peptide nucleic acid (PNAs) are the synthetic analogs of DNA. In this deoxyribose phosphate backbone supporting the nucleic acid bases are replaced by a noncharged peptide backbone. (1) PNA- FISH assay is successfully carried out using chromosome specific PNA probes on human lymphocytes, amniocytes as well as on isolated blastomeres. The direct chromosomal analysis is very important in studying the occurrence and etiology of chromosomal abnormalities in variety of clinical conditions. There are various limitations in the use of conventional FISH technique on this biological material. So this alternative of PNA technology is useful in detecting any chromosomal abnormalities in the sperm nuclei.

4) In ecology
FISH has been used extensively in identification and tracking organisms directly from the environment.

• Microbial activity in the open oceans.
FISH has been used to measure the amount of SAR 11 present in the ocean at 3,000m depth as well as in coastal waters and also in fresh waters. SAR11 bacteria has found to constitute 25 to 50% of the total population of the surface waters of the open oceans and nearshore waters. They are difficult to grown in the laboratory as they need low nutrient content like the oligotrophic oceans. SAR11 isolates stop replicating once the reach a density of 108 cells per milliliter.

• To study Crenarchaeota from nonthermal habitats.
Crenarchaeota can be studied from oxic marine waters worldwide by using fluorescent phylogenetic probes i.e. FISH.

• To identify and track the microbial population
FISH can be used to determine the percentage of a particular microbial population in a given sample. For example, to know the enumerate the number of Archaea in a given sample.
• To track bacteria and Archaea in pelagic waters.
There is a decrease in abundance of prokaryotic cells in the open ocean. The cell number is approximately 106 calls per ml. FISH technology has been used to track the distribution of bacteria and archaea in pelagic waters.

• To study the number and location of native bacteria on field grown wheat roots.
The native bacteria i.e. the filamentous bacteria and Pseudomonas spp. can be quantified and localized on the wheat roots grown in the field by using FISH. This data helps us to known the key areas which can be regulated in the rhizosphere to manage a sustainable agricultural system and the distribution of beneficial organisms.

5) In food industry
• To detect presence of Pseudomonas spp. in milk.
Pseudomonas species are the main spoilage causing organisms that lead to biological contamination of milk leading to adverse impact on quality, shelf life and consumer safety of milk. FISH probes specific to detect Pseudomonas spp. are used. This helps in simultaneous detection, enumeration and identification of Pseudomonas in milk. This rapid and accurate detection helps in identifying the source of contamination in the dairy plant, accurate test to validate the pasteurization treatment and prediction of the shelf life of the processed milk.

• To enumerate the number of Enterobacteriaceae in food sample.
Enterobacteriaceae is a marker organism indicating that the food is contaminated. FISH can be used to detect and enumerate the cultivable organism in food within one working day following a short cultivation step.

• Detection of Salmonella spp. and Listeria monocytogenes in complex food matrices.
The traditional method to detect Salmonella spp. and Listeria monocytogenes in food has numerous steps from pre-enrichment, enrichment, selective plating, identification and confirmation. FISH based methods helps in rapid identification and characterization of the organisms in the complex food matrices.

6) In forensics
• Gender identification of human hair.
The determination of sex from the hair sample can be done based on the presence or absence of sex chromatin in the cells of the root hair. FISH uses nonradioactive fluorescent labelled chromosome specific DNA probes that can rapidly identify the presence of the chromosome or the chromosome region in the cells. FISH is also used to detect the presence of male epithelial cells in cervicovaginal smears obtained from alleged rape cases.

• Identification of male epithelial cells in routine postcoital cervicovaginal smears.
FISH helps to identify sperm and non-sperm male cells in the cervicovaginal smears from the sex assault cases. Y chromatin specific DNA probes are used for identification.

7) In plant cytogenetic and genome research
• Chromosome synteny and evolution
The traditional method to study synteny and evolution is by using comparative genetic linkage mapping but this is very time-consuming and relies on established mapping population. FISH has proved to be a very good alternative to study the synteny and evolution pattern. FISH is a highly complementary to linkage mapping since it doesn’t require a population and also it can be done in a relatively less amount of time. FISH mapping has helped us to study evolution of several known translocations within the genome of chromosome of wheat and detect several new gene rearrangements in the wild type species.
• Gene duplication and amplification
The most common molecular mechanism for adaptation to the changing environment is gene duplication. The unequal crossover results in tandem duplication. Using the traditional molecular methods it is very difficult to characterize complex or massive duplications. FISH can be used to analyze such duplications.

FISH is a visually appealing technique and its uniqueness is that it can provide an intermediate degree of resolution between the DNA analysis and chromosomal investigation while retaining information at the single-cell level. Due to its accuracy it is extensively used in many fields and with minor modifications the procedure we can get greater sensitivity, specificity and resolution.

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