Lect 02 2025 PDF - Cytogenetics & CGH

https://www.slideshare.net/DrAkshayJoshi/aj-seminar-crd Lecture 2 MBB 438/738 2025 1 Lecture 2 MBB 438/738 2025 2  Molecular cytogenetics Traditional cytogenetics – smallest detectable change in size ~4Mb of DNA Molecular cytogenetics/FISH...

https://www.slideshare.net/DrAkshayJoshi/aj-seminar-crd Lecture 2 MBB 438/738 2025 1 Lecture 2 MBB 438/738 2025 2  Molecular cytogenetics Traditional cytogenetics – smallest detectable change in size ~4Mb of DNA Molecular cytogenetics/FISH – allows even the smallest changes to be detected Chromosome FISH (fluorescence in situ hybridization) – DNA probe is made with fluorescent tag – Rapid detection – Multiple imaging possibilities allow numerous probes to be used simultaneously Chromosome painting – Probes made of pools of DNA – Very useful for rearrangements Lecture 2 MBB 438/738 2025 3  replicated sister chromatids McNeil and Ried (2000) Lecture 2 MBB 438/738 2025 4  FISH Lecture 2 MBB 438/738 2025 5  Probes Lecture 2 MBB 438/738 2025 6  Chromosome FISH and painting methods Key here: these are patient chromosomes Lecture 2 MBB 438/738 2025 8 Single gene FISH to detect presence of gene DNA sequence (in this case 2 genes) Lecture 2 MBB 438/738 2025 9  Chromosome painting: An abnormal X chromosome (red) with additional material from chromosome 4 (green) Lecture 2 MBB 438/738 2025 10  Traditional Karyotyping Lecture 2 MBB 438/738 2025 11  Molecular karyotyping with the SKY (spectral karyotyping) procedure Unique probes exist for each chromosome Multiplex FISH Specialized software is used to interpret the fluorescent signal Can look at rearrangements very easily Lecture 2 MBB 438/738 2025 12  fluorescent hybridization SKY probes are then combined with and hybridized to metaphase DNA samples are labeled with chromosomes on a slide combinations of fluorochromes that produce a unique color for each chromosome = this gives rise to chromosome-specific probes Human Cot-1 DNA is commonly used to block nonspecific hybridization in microarray screening. It is placental DNA that is predominantly 50 to 300 bp in size and enriched for repetitive DNA sequences Lecture 2 MBB 438/738 2025 13  M Lecture 2 MBB 438/738 2025 14  G banding (A), actual SKY result (1) and computer image (2) Lecture 2 MBB 438/738 2025 15  SKY from cancer cells https://journals.plos.org/plosone/article/figure?id=10.1371/journal.pone.0160901.g001 Lecture 2 MBB 438/738 2025 16  Comparative Genome Hybridization Genetic alternations such as amplifications and deletions frequently contribute to tumorigenesis. These alternations changes the level of gene expression which modify normal growth control and survival pathways. Characterization of these DNA copy-number changes is important for both the basic understanding of cancer and its diagnosis. Comparative genomic hybridization (CGH) was developed to survey DNA copy-number variations across a whole genome. Lecture 2 MBB 438/738 2025 17 Comparative Genome Hybridization (CGH) using whole genomic DNA probes from different samples to examine chromosome changes in disease samples Start with DNA samples from a control (reference) and test sample (patient, tumour), make differently labelled probes from the DNA and compare them Key here: these probe sets are hybridized to NORMAL chromosomes Lecture 2 MBB 438/738 2025 18 Hybridization to NORMAL DNA is a key to this analysis The control/reference DNA probe should match perfectly and will hybridize uniformly along these chromosomes at the appropriate chromosomal location The test/patient DNA may contain losses of DNA, which means those parts of the genome would not have any corresponding green probe, so that you would only see the normal red signal on the slide. In contrast, if the test/patient DNA has any DNA duplications, then there would be more green probe hybridized to those sections of the genome. Lecture 2 MBB 438/738 2025 19 In regions of the genome where the test and control DNA are both normal, then you will see green and red overlap = yellow Quantification of probe intensity along length of chromosome Lecture 2 MBB 438/738 2025 20  Loss of DNA in test sample shifts colour to red Gain of DNA in test sample shifts colour to green Lecture 2 MBB 438/738 2025 21  Tumour DNA= green Control DNA = red merge Lecture 2 MBB 438/738 2025 22  Control DNA = red Tumour DNA= green Grey boxes are rich in heterochromatin and can’t be interpreted Loss of DNA in tumour sample Lecture 2 MBB 438/738 2025 23  Micro Array (or Matrix) CGH Uses plates containing fixed DNA samples Each spot corresponds to a unique position in the genome Basic Principles of Array CGH https://www.youtube.com/watch?v=MMkYSWzOwrY Lecture 2 MBB 438/738 2025 24  When samples are the same https://www.google.com/url?sa=t&rct=j&q=&esrc=s&source=web&cd=&cad=rja&uact=8&ved=2ahUKEwj_zvme-bH1AhVBKDQIHZplB9YQ- 4ACegQICxAH&url=https%3A%2F%2Fwww.youtube.com%2Fwatch%3Fv%3DMMkYSWzOwrY&usg=AOvVaw26pwoE11sdXLwO97HjuBV4 Lecture 2 MBB 438/738 2025 25 When test sample has aberrations Lecture 2 MBB 438/738 2025 26  aCGH results relative to all chromosomes Lecture 2 MBB 438/738 2025 27 CGH summary: DIM = diminished (less DNA) ENH = enhanced (more DNA) Array CGH Lecture 2 MBB 438/738 2025 28  CGH versus SKY Lecture 2 MBB 438/738 2025 29