BIO 150_Group 4_FISH.pdf

Bio 150 Introduction to Cellular and Molecular Biology Date Submitted: 07/13/23 FLUORESCENCE IN-SITU HYBRIDIZATION Score: Midyear AY 2022-2023 GUTIERREZ, LAGRASON, LAGUNDA, SANDAGA, TAGARINO I. INTRODUCTION Fluorescence In Situ Hybridization (FISH) is a molecular cytogenetic technique that dete...

Bio 150 Introduction to Cellular and Molecular Biology Date Submitted: 07/13/23 FLUORESCENCE IN-SITU HYBRIDIZATION Score: Midyear AY 2022-2023 GUTIERREZ, LAGRASON, LAGUNDA, SANDAGA, TAGARINO I. INTRODUCTION Fluorescence In Situ Hybridization (FISH) is a molecular cytogenetic technique that detects and locates a specific DNA sequence or an entire chromosome in a cell (Dutra, 2023). The general principle of FISH is based on fluorescent probes that bind to specific regions on the chromosome with a high degree of sequence complementarity. This can either be done with a short double-stranded RNA probe or a short double-stranded DNA probe. FISH has various applications, including disease diagnosis, gene mapping, the localization of mutations on chromosomes, and the identification of chromosomal abnormalities. The technique can also serve as a reference for comparisons among chromosomal arrangements of genes in related species (Shakoori, 2017). Joseph Gall and Mary Lou Pardue developed the FISH technique in the 1960s. It originally used radioactive labels in hybridization probes, and the sites were detected using autoradiography. However, it was later replaced with fluorescent labels because of their greater safety, stability, and ease of detection (Shakoori, 2017). DNA is made of two linked strands of molecules that are coiled together into a structure known as a double helix. Each strand is able to bind together because of the hydrogen bond present between its bases, where each sequence encodes specific biological information (Bates, 2023). Two complementary sequences then bind together or hybridize. The FISH technique makes use of the ability of one DNA strand to hybridize specifically with another DNA strand. It uses small DNA or RNA strands called probes that are attached to a fluorescent reporter molecule (Shakoori, 2017). The probes are complementary to specific parts of a chromosome or to a specific target sequence of sample DNA. Figure 1. Fluorescent in situ hybridization (FISH) identification of human chromosomes through chromosome painting. Image retrieved from Shakoori (2017). Figure 1 shows a computer-generated “false color” image of a human karyotype where the variations in fluorescence wavelength among the probes are enhanced to appear as distinct primary colors. The DNA probes that are specific to regions of particular chromosomes are attached to fluorescent markers, which are then hybridized with a chromosome spread. The unique pattern on each chromosome allows for the detection of chromosomal variations (Shakoori, 2017). II. METHODOLOGY Using heat, FISH unwinds the double helix structure to allow the binding of the probes to their complementary sequence in the patient’s DNA. If a small deletion is present in the region complementary to the probe, the probe will not be able to hybridize. If duplication is present, more probes will be able to hybridize (Shakoori, 2017). Fluorescence in-situ hybridization (FISH) uses complementary probes to bind and visualize specific nucleic acid sequences in cells or tissues. The probes are labeled with fluorescent molecules and applied to fixed samples. After hybridization and washing, fluorescence microscopy is used to visualize the bound probes, providing information about the abundance of the target sequences. location and Cell or tissue samples are obtained and fixed with 4% paraformaldehyde at room temperature for 10 minutes to preserve the cellular structures as well as maintain nucleic acid integrity (Dutra, 2023). The specific DNA or RNA probes were designed using bioinformatic tools (Hertoghs et al., 2003) to target complementary sequences of the nucleic acid of interest. The following reagents were used for probe design and labeling: DNA or RNA probes specific to the target sequence, fluorescently labeled nucleotides (e.g., Cy3-dUTP or FITC-dUTP), and labeling enzymes (e.g., Klenow DNA polymerase or reverse transcriptase) (Heslop-Harrison et al., 1991). After the samples were prepared, they were denatured at 75°C for 10 minutes to allow binding to the target nucleic acids. These samples were then mixed with the labeled probes. Hybridization was carried out at an appropriate temperature of 37°C overnight to facilitate specific binding between the probe and the target sequences. Hybridization buffers containing formamide (Sigma-Aldrich) were used to optimize the hybridization conditions. Following hybridization, samples underwent a series of post-hybridization washes to remove unbound or non-specifically bound probes. Stringent washing conditions were employed, including a wash with 0.2× SSC (Saline-Sodium Citrate) buffer at 60°C for 15 minutes, to ensure specific hybridization and reduce background noise (O’Connor, 2008). Finally, to visualize cellular structures and enhance contrast, samples were counterstained with appropriate dyes or stains, such as DAPI (4',6-diamidino-2-phenylindole, Thermo Fisher Scientific) for nuclear staining. Mounted samples were protected using an appropriate mounting medium to preserve fluorescence signals and prevent photobleaching. IV. DISCUSSION How are probes made? Fluorescence in situ hybridization probes are short DNA or RNA sequences that are labeled with fluorescent molecules. These are made to complement target sequences, therefore, only allowing them to GUTIERREZ, LAGRASON, LAGUNDA SANDAGA, TAGARINO specifically bind to these regions. Through hybridization to a complementary target sequence, FISH probes allow for its visualization under a fluorescence microscope. These probes are classified into different types: gene specific, centromeric, whole chromosome, telomeric probes. Each of the probe types mentioned target a specific location, and acts on a variety of applications (Ford & Reid, 2000). FISH probes can be labeled with different fluorescent molecules such as fluorescein isothiocyanate, cyanine dyes, or Alexa Fluor dyes. The fluorophore used could vary depending on the requirements of the procedure to be conducted (Lukumbuzya et al., 2019). As discussed, one crucial step in Fluorescent In-Situ Hybridization is the probe design. The probe is the most critical component in ensuring the specificity of hybridization. Essentially, commercially available probes are best used in facilitating this assay. However, when synthesizing probes, pure DNA is required and proper quality control must be performed. Techniques such as gel electrophoresis or spectroscopy may be employed to evaluate the probe’s integrity and fluorescence. It is also important to note that Cot-1 DNA is supplemented along with the probes. Cot-1 DNA is typically used in FISH procedures to act as a blocking agent to minimize non-specific binding of probe sequences. By supplementing the probe with Cot DNA, “background noise” is reduced which allows for a more specific and accurate detection of the target sequences (Wang et al., 1995). With the DNAse enzyme, random cuts in the DNA are made which are called nicks. The DNAse is an endonuclease which catalyzes the cleavage of DNA molecules within the polynucleotide chain by targeting the phosphodiester bonds in between them. Then, DNA polymerase attaches special nucleotides with fluorophore (fluorescein, Cy3, or Cy5) to the DNA; this is to enable visualization and detection. Lastly, Ligase enzyme seals the nicks with the fluorescent nucleotides (Bartlett, 2004). What did this tell us about chromosome organization? The fluorescent in-situ hybridization, as discussed, uses labeled fluorescent probes which consist of nucleotide base sequences complementary to that of the target DNA sequence with an attached fluorescent marker, that is hybridized to the DNA and serves as colored markers for the particular DNA sequence under a fluorescent microscope. This has been utilized Group 4 together with karyotyping to observe chromosome organization by determining the location of the corresponding genes that are expressed or responsible for certain characteristics. This has enabled the diagnosis of chromosomal abnormalities such as gene translocation, deletion, and/or duplication (O’Connor, 2008). In relation to this there are different probes utilized to achieve these observations, namely: locus specific probes, alphoid ro centromeric repeat probes and whole chromosome probes. The locus specific probes are used for determining gene location within the chromosome and number of its copies while alphoid or centromeric repeat probes are used to determine if an individual has correct number of chromosomes and lastly, whole chromosome probes which used to generate spectral karyotype which is useful for detecting chromosomal abnormalities (Shakoori, 2017). One illustration of the application of FISH is in the diagnosis of Charcot-Marie-Tooth type 1A, a neurological condition caused by gene duplication on chromosome 17. Red probes were used to locate the duplicated gene while a green probe was used in the determination of the chromosome 17. single nearby red signal. Thus, indicating duplication of the gene in chromosome 17 (O’Connor, 2008). The observations of the changes in chromosome organization with FISH implies that chromosomes have a certain degree of specificity and that the localization and number of genes present in chromosomes have a significant effect on gene expression, as reflected by significant changes in an organism’s anatomy and/or physiology; thus, the chromosome organization has an implication on the regulation of DNA processes (Zimmer and Fabre, 2011). V. REFERENCES Journals: Bartlett, J.G. (2004). Fluorescence In Situ Hybridbization: Technical Overview. In Molecular Diagnosis of Cancer (pp.77-88). https://doi.org/10.1385/1-59259-760-2:077 Ford, N.M. & Ried, T. (2000). Novel molecular cytogenetic techniques for identifying complex chromosomal rearrangements: Technology and applications in molecular medicine. Expert reviews in molecular medicine. 2000. 1-14. Hertoghs, K.M.L., Ellis, J.H. and Catchpole, I.R (2003) Use of locked nucleic acid oligonucleotides to add functionality to plasmid DNA. Nucl. Acids Res.31 (20): 5817-5830. Heslop-Harrison J, Schwazarcher T, Anamthawat-Jónsson K, Leitch AR, Shi M. 1991. In situ hybridization with automated chromosome denaturation. Technique. 3109-15. Lukumbuzya, M., Schmid, M., Pjevac, P., & Daims, H. (2019). A Multicolor Fluorescence in situ Hybridization Approach Using an Extended Set of Fluorophores to Visualize Microorganisms. Frontiers in Microbiology, 10. https://doi.org/10.3389/fmicb.2019.01383 Figure 2. Fluorescent in situ hybridization (FISH) identification of human chromosomes 17 in Charcot-Marie-Tooth disease Type 1A. As shown in Figure 2, it was determined that there was duplication of the gene in one of chromosome 17 dictated by the two red signals near the green signal while the other green signal only has a GUTIERREZ, LAGRASON, LAGUNDA SANDAGA, TAGARINO O'Connor, C. (2008). Fluorescence in situ hybridization (FISH). Nature Education, 1(1), 171. Wang, Y., Minoshima, S., & Shimizu, N. (1995). Cot-1 banding of human chromosomes using fluorescence in situ hybridization with Cy3 Group 4 labeling. Japanese Journal of Human Genetics, 40(3), 243–252. https://doi.org/10.1007/bf01876182 Online Articles: Bates, S. A. (2023, July 10). Deoxyribonucleic Acid (DNA). National Human Genome Research Institute. Retrieved July 12, 2023, from https://www.genome.gov/genetics-glossary/De oxyribonucleic-Acid Dutra, A. (2023). FLUORESCENCE IN SITU HYBRIDIZATION (FISH). National Human Genome Research Institute. Retrieved from https://www.genome.gov/genetics-glossary/Flu orescence-In-Situ-Hybridization Shakoori, A. R. (2017). Fluorescent In Situ Hybridization (FISH). And Its Applications. PubMed Central. Retrieved from https://www.ncbi.nlm.nih.gov/pmc/articles/PM C7122835/#Sec4title Zimmer, C. and Fabre, E. (2011). Principles of chromosomal organization:lesson from yeast. PubMed Central. Retrieved from https://www.ncbi.nlm.nih.gov/pmc/articles/PM C3051815/ GUTIERREZ, LAGRASON, LAGUNDA SANDAGA, TAGARINO Group 4