Biological And Chemical Analytics (BIOT303) Lecture Notes PDF

Biological and chemical Analytics (BIOT303) Faculty of Biotechnology Biological and Chemical Analytics Lecture 9: Molecular Cytogenetic Analysis Dr. Rana A. Youness Head of M...

Biological and chemical Analytics (BIOT303) Faculty of Biotechnology Biological and Chemical Analytics Lecture 9: Molecular Cytogenetic Analysis Dr. Rana A. Youness Head of Molecular Genetics Research Team (MGRT) E-mail: [email protected] Office: S1-610 Office Hours: Thursday 12-1 pm Intended Learning Outcomes (ILOs): By the end of the lecture and after reading the appropriate text books, the student should be able to understand: Define Clinical and Molecular Cytogenetics Understand the procedure of karyotyping Recall sources of specimens for chromosomal analysis Understand the principle of Fluorescence in situ Hybridization (FISH) Understand the principle of Multiplex-FISH, Spectral Karyotyping (SKY) Understand the principle of Array-based Comparative genomic hybridization (aCGH) 2  What is Clinical Cytogenetics? Definition: It is the study of the relationship of chromosomal alterations (including both structural & numerical) and genetic disease in humans using the conventional techniques 3  What is Molecular Cytogenetics? Definition: It is a study of genetic disorders associated with chromosomal abnormalities using new technologies that combine cytogenetic and molecular techniques 4  Karyotyping procedure (a) Culture blood cells (b) Spin down cells in a Add hypotonic salt (c) Add fixative (lymphocytes): add colchicine centrifuge; discard solution and (preservative) to cell to stop mitosis at metaphase supernatant resuspend the cells suspension; dry and stain 5 with Giemsa  Human Karyotype But How? Who sort them? Male Female  Ideogram Ideogram: It is a diagrammatic representation of the chromosomes showing their relative size and chromosomal landmarks  Ideogram of Chromosomes Metacentric Submetacentric Acrocentric (Chromosome 1) (Chromosome 9) (Chromosome 14) Euchromatin and heterochromatin Heterochromatin is a highly condensed, mostly highly repetitive DNA arranged in tandem Euchromatin is arrays, very few a lightly packed genes form of chromatin that is enriched in genes, and is often (but not always) under active transcription 9 Sources of specimens for chromosome analysis 10  Molecular cytogenetic techniques 1. Fluorescence in situ Hybridization (FISH) 2. Multiplex-FISH, Spectral Karyotyping (SKY) 3. Array-based Comparative genomic hybridization (aCGH)  1- Fluorescence in situ hybridisation (FISH) o Maps DNA sequences to specific regions of human chromosomes o FISH involves the use of fluorescently labelled DNA probes that hybridise to complementary chromosomal regions o The net result is a fluorescent dot at the chromosomal location where the labelled probe binds o FISH allows a higher level of resolution than standard G-banding approaches  1- Fluorescence in situ hybridisation (FISH) oFISH allows detection and localization of specific DNA sequences in the interphase or during the metaphase oPros: results are available within 24- 48 hrs oCons: it fails to detect big structural rearrangements, some abnormalities are undetectable and sometimes the procedure can destroy the test itself  Interphase-FISH Interphase-FISH: blastomeres from PGD 150 kb (maternal & paternal chr. 1) Rainbow-coloured chromosome 2- Multiplex-FISH or Spectral Karyotyping (SKY) 15  2- Multiplex-FISH or Spectral Karyotyping (SKY) Multiplex-FISH or spectral karyotyping is a laboratory technique that allows scientists to visualize all of the human chromosomes at one time by painting each pair of chromosomes in a different fluorescent colour Remember: with the traditional/conventional karyotyping, the whole set of chromosomes is seen in black and white Interpreting G-banding karyotype requires an expert and might need hours to examine a single chromosome ☺ By using SKY though, even non-experts can easily detect if there are any abnormality where a chromosome painted in 1 colour has a small piece of a different chromosome painted in another colour attached to it  SKY involves preparation of a large collection of short sequences of single stranded DNA (probes) Each of the individual probes is complementary to a unique region of one chromosome 16  2- Multiplex-FISH or Spectral Karyotyping (SKY) All of probes make up a collection of DNA that is complementary to all of the chromosomes within the human genome Each probe is labelled with a fluorescent colour and is designated for a specific chromosome. For e.g.: probes for chromosome 1 are labelled with yellow, those for chromosome 2 are labelled with red and so on When these probes are mixed with the human chromosome in a human cell, the probes hybridize to the DNA in chromosome As they hybridize, the fluorescent probe essentially paints the full set of chromosomes in a rainbow colour Each chromosome has a completely different colour We can use our computers to analyse the painted chromosomes to determine whether any of them exhibited translocation or any other structural abnormalities We can also use different colours to paint the different binding pattern on its chromosome 17 2- Multiplex-FISH or Spectral Karyotyping (SKY) 18  Comparative Genomic Hybridisation (CGH) The technique requires long time and very expensive equipment Total DNA from control cells (labelled with green dye) and total DNA from test samples (labelled with red dye) DNA from both sources will be denatured and co-hybridized Chromosomal regions that are equally represented in test and control samples will appear yellow Regions of DNA gain are red, while that of DNA loss appear green Slide is then placed in a specific scanner connected to a computer 19 1 million probes & 1 400,000 probes 180,000 probes 60,000 probes & position on which only 1 & 2 positions & 4 positions 8 positions for 8 sample can be loaded for 2 samples for 4 samples samples  MCQ Fluorescent in situ hybridization (FISH) a) requires deoxynucleotides b) requires a labeled probe c) is used in genetic mapping of genomes d) requires a DNA polymerase b) requires a labeled probe 24