MLT552 Lecture 11 Molecular Cytogenetics 2024 PDF

MLT552: MOLECULAR DIAGNOSTICS & CYTOGENETICS LECTURE 11: MOLECULAR CYTOGENETICS Ts. DR. MOHD FADLY MD AHID 31st DECEMBER 2024 LEARNING OUTCOMES At the end of this lesson, you should be able to: ▪ D...

MLT552: MOLECULAR DIAGNOSTICS & CYTOGENETICS LECTURE 11: MOLECULAR CYTOGENETICS Ts. DR. MOHD FADLY MD AHID 31st DECEMBER 2024 LEARNING OUTCOMES At the end of this lesson, you should be able to: ▪ Discuss the limitations of conventional cytogenetic technique. ▪ List the type of molecular cytogenetic techniques. ▪ Describe the principle, procedures and significance of molecular cytogenetic techniques. 2 Conventional Cytogenetics 3  Time-consuming Require a high rate of dividing cells (3 – 7 days) with good chromosomal morphology Sensitivity of chromosomal banding techniques is limited LIMITATIONS OF CONVENTIONAL CYTOGENETICS Chromosomal changes in leukemic cells are very Expert technician is required often complex or could be cryptic under the limit of to arrange chromosome light microscopy – result in a false negative result 4  MOLECULAR CYTOGENETIC TECHNIQUES Fluorescence in situ Comparative Genomic Hybridization (FISH) Hybridization (CGH) Multicolour FISH New technologies that combine cytogenetic and molecular techniques. Provide a way to detect complicated, cryptic & submicroscopic rearrangements that remain undetected by conventional cytogenetic analysis (G-banded karyotyping). 5 Fluorescence in situ Hybridization (FISH) ▪ Principle: To detect & localize the presence or absence of specific DNA sequences on chromosome. FISH allows for the study of genetic abnormalities that are too small to visualize by conventional cytogenetic technique and too large to detect using standard DNA sequencing. Use of fluorescently-labelled DNA probes that bind onto a high complementary part (target DNA sequences) of a chromosome on a microscope slide & visualized using a fluorescence microscope. 6 Fluorescence in situ Hybridization (FISH) ▪ Applications: Gene mapping Total chromosome analysis Chromosome identification Translocation analysis Aneuploidy detection Unique sequence detection Sexing for X-linked diseases Microdeletion syndrome analysis Marker chromosome identification Gene amplification analysis 7 Fluorescence in situ Hybridization (FISH) ▪ Procedures: FISH can be performed on previously prepared metaphase spreads or on nondividing cells (interphase nuclei) from fresh samples or formalin fixed tissues preparations. The procedure involves: Step I: Denaturation – conversion of dsDNA into ssDNA Step II: Hybridization – binding of DNA probe to target DNA Step III: Post-hybridization washing – washing of unbound DNA probe Step IV: Counter stain – staining of cytoplasm/background Step V: Visualization – using fluorescence microscope 8 Fluorescence in situ Hybridization (FISH) Hybridization steps 9 Fluorescence in situ Hybridization (FISH) ▪ FISH probe – a synthesized DNA fragment of variable length (100 – 1000 bases long) labelled with a molecular marker of either radioactive or fluorescence molecules ▪ Types of FISH probe: 1. Centromere-specific probe 2. Whole chromosome (‘painting’) probe 3. Single-copy (locus-specific) probe 10 Fluorescence in situ Hybridization (FISH) 1. Centromere-specific probe ▪ centromere enumerating probe (CEP) ▪ repetitive sequence probes that target highly repetitive 171 kb α-satellite sequences of centromeric DNA ▪ α-satellite sequences of chromosomes differ Monosomy 7 which allow detection of specific chromosomes ▪ useful for detection of numerical abnormalities (aneuploidy) in both metaphase and interphase cells Trisomy 8 11 Fluorescence in situ Hybridization (FISH) 2. Whole chromosome probe ▪ chromosome ‘painting’ probe (WCP) for metaphase only ▪ consist of numerous overlapping probes that recognise and bind to specific non-repetitive DNA sequences along the entire length of individual (targeted) chromosomes ▪ can be used to identify marker chromosomes (rearranged chromosomes of unknown origin) or translocations that are otherwise not evident or difficult to interpret with routine banding cytogenetics 12 Fluorescence in situ Hybridization (FISH) 3. Single-copy (locus-specific) probe ▪ most widely used type of FISH probe ▪ unique sequence probe , range in size from approximately 1 kilobase (Kb) to >1 megabase (Mb) that target distinct chromosomal regions t(9;22) (locus) ▪ used to detect the presence of loss or amplification (increased number of copies of a specific locus), or to detect the presence of rearrangement (structural abnormalities) 13 Multicolour FISH ▪ Multicolour FISH, multiplex FISH (M-FISH) or spectral karyotyping (SKY) ▪ a 24-colour, multichromosomal painting technique that allows for the simultaneous visualization of all chromosomes of a metaphase in a single hybridization step ▪ useful in evaluation of cases with complex chromosomal abnormalities, which often preclude an accurate interpretation by conventional cytogenetics 14 Comparative Genomic Hybridization (CGH) ▪ A technique based on the comparison of two genomic DNA populations, a specimen (test) DNA and a reference (normal) DNA. ▪ Differentially labelled test DNA from a specimen (test) and reference (normal) DNA are hybridized simultaneously onto normal human metaphase chromosome spreads, competing for the same targets. ▪ The hybridization products are detected with two different fluorochromes (usually red and green). The fluorescence signal (ratio of the green and red fluorescence) of the test sample is compared to that of a normal sample. 15 Comparative Genomic Hybridization (CGH) ▪ Changes in the ratio of the intensities of the two fluorochromes as compared to the expected one-to-one ratio seen in the control sample indicates regions of gain or loss of DNA sequences, such as deletions, duplications, or amplifications. ▪ Areas with a green to red ratio >1 indicate amplifications, a value