Spectral Karyotype[SKY-Rupendra Shrestha]

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Spectral Karyotype:

S p e c t r a l K a r y o t y p e Rupendra Shrestha Clinical Molecular Geneticist Department of Medical Genetics, Manipal Hospital, Bangalore, India

Spectral Karyotype :

Spectral Karyotype Spectral karyotyping is a molecular cytogenetic technique used to simultaneously visualize all the pairs of chromosomes in an organism in different colors. The SKY technique is useful for identifying chromosome abnormalities . Clinical Molecular Geneticist/Manipal Hospital/Rupendra Shrestha

Spectral karyotype:

Spectral karyotype Clinical Molecular Geneticist/Manipal Hospital/Rupendra Shrestha Fluorescently labeled probes for each chromosome are made by labeling chromosome-specific DNA with different fluorophores . C ombinatorial labeling method is used to generate many different colors.( limited number of spectrally-distinct fluorophores) Spectral differences generated by combinatorial labeling are captured and analyzed by using an interferometer attached to a fluorescence microscope. Image processing software then assigns a pseudo color to each spectrally different combination, allowing the visualization of the individually colored chromosomes

Spectral karyotype:

Spectral karyotype Clinical Molecular Geneticist/Manipal Hospital/Rupendra Shrestha

Cytogenetic analysis using spectral karyotype:

Cytogenetic analysis using spectral karyotype Clinical Molecular Geneticist/Manipal Hospital/Rupendra Shrestha

STEPS INVOLVED IN SKY:

STEPS INVOLVED IN SKY Probe labeling Chromosome preparation from tissue. Pre-treatment and denaturation. Probe denaturation. Hybridization. Post hybridization washes and detection Image acquisition Hybridization of SKY probe to previously G-banded slides. Clinical Molecular Geneticist/Manipal Hospital/Rupendra Shrestha

Probe labelling:

Probe labelling SKY probes are commercially available created from flow-sorted chromosomes that are usually amplified and labelled using DOP-PCR 24-chromosome probe cocktail is generated by the combination of 5 pure dyes. This allows 2 n-1 or 31 combinations. The color separation for each chromosome is based on individual spectral properties of each fluorochrome as detected by a specially created triple filter ( SKYCube ™ ) during a single acquisition step Clinical Molecular Geneticist/Manipal Hospital/Rupendra Shrestha

Chromosome Preparation from Tissues:

Chromosome Preparation from Tissues C linical and research molecular cytogenetic laboratories ability to hybridize and detect all the chromosomes in the genome. cytogenetic preparations sample(hematopoietic sources or solid tissues), the cells used must be mitotically active so that adequate good quality metaphases can be generated after short-term tissue culture. colcemid prevent spindle formation and keep cells at metaphase. proper colcemid times and concentration is maintained. Clinical Molecular Geneticist/Manipal Hospital/Rupendra Shrestha

Chromosome Preparation from Tissues:

Chromosome Preparation from Tissues Cell fixation also involves hypotonically Swelling the cells, then slowly introducing a methanol:acetic acid fixative . Careful Attention to the hypotonic treatment and first methanol: acetic acid fixation can yield Cell suspensions that are free from cellular and cytoplasmic debris . Drying environment during slide making and age of the slide (affects quality of chromosomal preparation). Humidity and temperature will significantly influence the rate of fixative evaporation during slide making. slide making is performed near a steam source or humidifier (humidity less than 30%). Clinical Molecular Geneticist/Manipal Hospital/Rupendra Shrestha

Pre-treatment and Denaturation:

Pre-treatment and Denaturation Once metaphase preparations have been made, the slides must be assessed to determine the necessary slide pre-treatment. All slides should be assessed by phase contrast microscopy before SKY analysis. Unavoidable background ---- due to presence of cellular and cytoplasmic debris ---- removed by protease treatments. after protease treatment, the DNA must be denatured to a single stranded state to hybridize to the labelled probe . Clinical Molecular Geneticist/Manipal Hospital/Rupendra Shrestha

Pre-treatment and Denaturation:

Pre-treatment and Denaturation H eat denaturation using a 70% formamide solution. Formamide lowers the melting temperature of DNA, thus a 75°C solution of formamide is sufficient to denature DNA into single strands . After denaturation of the target DNA, the slides must immediately be placed in 70% ethanol followed by an increasing alcohol series to keep the DNA target in its denatured state and to remove any residual moisture. The denaturation of the target DNA is the most critical step in any FISH-based assay. Too little denaturation causes poor hybridization efficiency and accessibility of the probe to the target DNA. Too much denaturation destroys the DNA also resulting in poor hybridization efficiency Clinical Molecular Geneticist/Manipal Hospital/Rupendra Shrestha

Probe Denaturation:

Probe Denaturation SKY probe, must also be denatured into its single stranded state in order for hybridization to occur. probe comes already resuspended in a hybridization solution usually containing formamide and dextran sulfate. F ormamide allows the DNA to melt at lower temperatures. The use of unlabelled COT-1 DNA in FISH probes serves to suppress repetitive sequence hybridization. Thus , the probe must first be heat denatured at 75°C, then allowed to pre-anneal at 37°C such that the COT-1 DNA can hybridize to and block background signals from these highly repetitive sequences. COT-1 DNA is the fraction of DNA sequences, which have the fastest annealing properties, the 1 h incubation at 37°C will not cause the re-annealing of the labelled DNA. Clinical Molecular Geneticist/Manipal Hospital/Rupendra Shrestha

Hybridization:

Hybridization Once both the target DNA on the slides and the DNA probe has been denatured, the probe is applied to the slides and allowed to hybridize together at 37°C for a minimum of 48 h. The signal strength following hybridization will depend primarily on the completeness of the denaturation process of the target DNA. In addition, the presence of cellular and cytoplasmic debris surrounding the chromosomes, can greatly impair the accessibility of the probe to the target chromosomes. Clinical Molecular Geneticist/Manipal Hospital/Rupendra Shrestha

Denaturation and Hybridization:

Denaturation and Hybridization Clinical Molecular Geneticist/Manipal Hospital/Rupendra Shrestha

Post-Hybridization Washes and Detection:

Post-Hybridization Washes and Detection post hybridization washes and detections to remove any unbound probes, a lower concentration of formamide at 45°C is used followed by a treatment with 1X SSC also at 45°C. blocking step is performed to reduce any background or noise present during the immunological detection steps to follow . blocking solutions ( combination of bovine serum albumin and a detergent in an isotonic buffer .) Clinical Molecular Geneticist/Manipal Hospital/Rupendra Shrestha

Post-Hybridization Washes and Detection:

Post-Hybridization Washes and Detection commercial SKY probes are combinations of specific paints in which some chromosomal paints have been directly labelled with a fluorochrome , and others have been indirectly labelled with a hapten. The detection reagents provided in the kit contain antibodies to indirectly detect labelled paints. Gentle agitation during these washes helps to reduce the background signals that may appear due to the presence of cellular and cytoplasmic debris. The final step is the counterstaining of the DNA using DAPI mixed with an antifading mounting medium. The slides can then be stored at –20° where the signals can last for several months. Clinical Molecular Geneticist/Manipal Hospital/Rupendra Shrestha

Counterstain :

Counterstain DAPI ( diamidino -2 – phenylindole ) Vysis Propidium iodide Clinical Molecular Geneticist/Manipal Hospital/Rupendra Shrestha

Image Acquisition:

Image Acquisition Image acquisition and analysis software play key role for the greatest success in applying SKY analysis to cytogenetic cases . the differentially labelled whole chromosome paints are distinguished from each other by their unique spectral properties . Using a fluorescent microscope equipped with the SKYCUBE, light passes through a Sagnac interferometer focussed on a charged-coupled device ( CCD) detector. Clinical Molecular Geneticist/Manipal Hospital/Rupendra Shrestha

Hybridization of SKY probes to Previously G-banded Slides: Slide Pre-treatment:

Hybridization of SKY probes to Previously G-banded Slides: Slide Pre-treatment Performance of SKY analysis on previously G-banded material for; To verify an inconclusive chromosomal aberration on a specific metaphase cell. In this situation an image of the G-banded metaphase cell of interest should be captured, the microscope coordinates noted , and promptly prepared for SKY analysis as soon as possible (i.e., within 2 wk ). If there are a limited number of slides remaining from the patient sample of interest, it is sometimes helpful to use a superfluous slide from another sample prepared the same week to derive the best conditions with respect to aging and storage. Clinical Molecular Geneticist/Manipal Hospital/Rupendra Shrestha

Spectral karyotype observation :

Spectral karyotype observation Clinical Molecular Geneticist/Manipal Hospital/Rupendra Shrestha

Limitation of sky :

Limitation of sky I nability to readily detect deletions or other intrachromosomal structural changes such as inversions. Clinical Molecular Geneticist/Manipal Hospital/Rupendra Shrestha

Genome-Wide Profiling: Karyotyping :

Genome-Wide Profiling: Karyotyping Spectral karyotyping is a method that allows researchers to use probes that assign a different color spectrum to each chromosome, making it possible to see where genetic material has been added or exchanged within a cancer genome Clinical Molecular Geneticist/Manipal Hospital/Rupendra Shrestha

Conventional Analysis Tools:

Clinical Molecular Geneticist/Manipal Hospital/Rupendra Shrestha Conventional Analysis Tools Karyotyping (G-banding) Provides a global view of metaphase chromosomal characteristics (number, type, shape etc ) Each chromosome has a characteristic banding pattern that helps to identify them Spectral Karyotyping (SKY) Allows simultaneous visualization all the chromosomal pairs in different colors using chromosome specific probes More accurate than G-banding Fluorescent in situ Hybridization (FISH) More specific and sensitive than karyotyping Uses fluorescent probes to detect and localize the presence or absence of specific DNA sequences on chromosomes Resolution: 5 Mb – Metaphase 2 Mb – Interphase 0.5 Mb – Fibre FISH

Comparison of cytogenetic techniques for identifying chromosomal abnormalities. :

Comparison of cytogenetic techniques for identifying chromosomal abnormalities. Clinical Molecular Geneticist/Manipal Hospital/Rupendra Shrestha

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Clinical Molecular Geneticist/Manipal Hospital/Rupendra Shrestha

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Clinical Molecular Geneticist/Manipal Hospital/Rupendra Shrestha Rupendra speaks;" More u read ,the more confusion; confusion triggers our curiosity, curiosity on new things is searching for it that’s called research; researcher are honoured as scientists that’s we should aim in life” THANK YOU FOR KIND ATTENTION

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Clinical Molecular Geneticist/Manipal Hospital/Rupendra Shrestha