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Chromosome painting

Chromosome painting. Presented By- Anamika Das Roll no-06 Msc Biotechnology Gauhati University. Introduction.

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Chromosome painting

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  1. Chromosome painting Presented By- Anamika Das Roll no-06 Msc Biotechnology Gauhati University

  2. Introduction • Chromosome-Painting, a term 1st used by pinkel et al in1983.Chromosome ‘painting’ refers to the hybridization of fluorescently labeled chromosome-specific, composite probe pools to cytological preparations. Chromosome painting allows the visualization of individual chromosomes in metaphase or interphasecells. • Chromosome painting coupled with FISH is now used to enhance the identification of chromosomal rearrangements,breakpoints and also the extra chromosomal material.

  3. Amplification of small numbers of flow sorted chromosomes by the PCR allows labelled chromosome paints to be generated in a matter of days. • These technologies have enabled the development of Reverse chromosome painting. • It is able to identify not only the chromosomal origin of marker chromosomes but also the regions and the breakpoints involved.

  4. Principle of chromosome-painting • The usual starting material for the production of chromosome painting is flow sorted chromosomes. • Short sequences from the sorted material are generated and amplified. • The PCR products are then labeled with a flurochrome or a hapten to generate a complex probe suitable for use in in-situ hybridisation.

  5. Hybridized onto metaphase spreads or interphasenuclei,duplexes form between complementary sequences in the chromosome painting and target material. • Visualization of the labeled DNA with fluorochrome or enzyme conjugated affinity reagent systems results in the painted chromosome being strikingly identified in metaphase spreads and interphase nuclei

  6. In its earlier stages this technique used composite probes for experiments that were recombinant libraries from specific chromosomes isolated by a method called flow cyclometry. • The painting probes, these days are generated by micro dissected chromosomes more effectively by a general PCR amplification of the chromosomal DNA with a degenerated primer.

  7. Aberrant chromosomes, are hybridized onto metaphase spreads from normal subjects which reveal about the chromosomal origin of the aberrant chromosome. • Chromosome painting is a special application of fluorescencein situ hybridization also called as FISH. • It uses DNA probes in which the starting DNA comprises of a large collection of different DNA fragments from a single type of chromosome

  8. As a result of this, the hybridization signal thus formed. • Initially only a small number of differently colored fluorescent dyescalled fluorophores were limited to chromosome painting. • But in order to increase the number of differentiating chromosomes by using only a limited number of fluorophores, two main approaches were opted.

  9. Combinatorial labeling • using a combination of different fluorophores. • The mixed color patterns thus generated are as such not detected by the standard fluorescence microscopy with appropriate filters.

  10. Rather a preferred way is using an automated digital image analysis for the various combinations of fluorophores which are then assigned some artificial pseudocolor. The 24 different human chromosomes simultaneous visualization was achieved using the following two approaches: • Multiplex FISH(M-FISH)- The approach as reported by Speicheret al. (1996) uses the digital images that are acquired separately for each of five different fluorophores using a CCD (charge coupled device) camera.

  11. Then these images were analyzed by a software package that generates a composite image where each chromosome has got a different pseudocolor as per the fluorophore composition it has. • Spectral karyotyping (SKY): This approach is reported by Schrock et al. (1996) where the CCD imaging has a combination of Fourier spectroscopy. The fluorescent spectral wavelengths for each pixel are then assessed by using an interferometer and this is assisted by a specific pseudocolor assigned by a computer program depending of the corresponding fluorescence spectrum being identified.

  12. With these techniques, chromosome painting has found its applications using marker chromosomes in clinical and cancer cytogenetics

  13. Applications of Chromosome Painting • To find out the location of a specific gene located on a specific chromosome. • Detection of translocation • Detecting chromosome abnormalities • To find out the chromosomal similarity between divergent species

  14. Clinical applications:-With the use of this technique, it is possible to easily identify the presence of numerous chromosomal translocations and unambiguously identify structural alterations in cancer cell lines.The application of these techniques should facilitate analysis of chromosomal aberrations and genetic abnormalities in various human diseases including cancer.

  15. Thank you….

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