Basic Cytogenetics Protocols PDF

For classroom use only Basic Cytogenetics Protocols The study of human chromosomes PREPARED BY: Christian Joseph N. Ong, MSc, LPT D e p a rtme nt o f Bi o l o g y C o l l e g e o f Sc i e n c e Bu l a c a n Sta te U n i ve r si ty...

For classroom use only Basic Cytogenetics Protocols The study of human chromosomes PREPARED BY: Christian Joseph N. Ong, MSc, LPT D e p a rtme nt o f Bi o l o g y C o l l e g e o f Sc i e n c e Bu l a c a n Sta te U n i ve r si ty 1 Tao Chiu Hsu ❑ 1917 – 2003 ❑ Chinese American Cell Biologist ❑ “Father of Mammalian Cytogenetics” ❑ 13th President of the American Society of Cell Biology ❑ Bleomycin Assay and Cancer Susceptibility 2 Cytogenetics ❖ The study of genetic phenomena through the analysis of chromosomes under the light or electron microscope. ❖ Structure, Behavior and Function of Chromosomes ❖ Chromosomes are the carrier of genes 3 Elements of Cytogenetics 1. Sample 2. Processing Method 3. Analysis 4 1.The Sample What biological materials (cells/tissues) are used in cytogenetic analysis? 1. Viable cells with nucleus ◦ Dead cells cannot be cultured ◦ Tissues that are formalinized cannot be cultured 1.2 Sterile/Aseptic condition ◦ Aseptic handling of extraction, transport and actual processing ◦ Contaminated cells cannot be cultured 5 2. The Processing of sample 1. Sample preparation 2. Actual Culturing Procedure 3. Harvesting and Fixation 4. Slide Preparation 5. Staining 6 2.1 Sample Preparation Requirement: Single cell suspension or ❑ Solid Tissues: ▪ Surgical resections (malignant tissues) ▪ Fetal tissues: (abortus material), chorionic villus ❑ Liquid samples/liquid biopsies ▪ Whole blood ▪ Bone marrow Aspirate (WBC) ▪ Amniotic Fluid ▪ Pleural effusion 7  Solid Tissue Preparation Mechanical Disaggregation Enzymatic disaggregation Prior Washing of samples Done to increase cell recovery ▪ Usually used in processing soft ▪ Use of enzymes such as trypsin or tissue samples (liver, spleen) collagenase ▪ Sample is cut into small pieces ▪ to digest proteins that hold cells using a scalpel, scissors together ▪ Cells are forced into a syringe and▪ Tissue is transferred a suitable needle or a series of sieves (mesh) growth medium for incubation 8  Explant culture (an alternative method for solid tissues) ▪ Culture of small pieces of tissue New growth of cells ▪ Surgically removed from animal tissue or organ Skin fragment ▪ Tissue sticks on the surface of flask or petri dish ▪ With complete media ▪ Maintenance of the original histotypic architecture and biochemical properties of the cells Explant culture of a skin (fragment). ▪ Closely resembles the tissue in Notice new outward growth of cells at vivo than established cell lines the periphery of the fragment. Gfycat.com 9 Liquid samples/biopsies ❑ Cells are separated from the liquid usually by centrifugation ▪ Pleural effusion (fluid around the lungs) ▪ Amniotic fluid ❑ Chromosome analysis of effusions is useful in the diagnosis of cancer. ❑ Conventional cytology has been reported to yield high percentage of false negatives ❑ Cytogenetic analyses of amniotic fluid are nearly 100% accurate for detecting large fetal chromosome abnormalities 10  Sampling of chorionic villi and amniocentesis ssl.adam.com; slideplayer.com 11  2.2 Actual culturing 1.Short Term Culture Whole blood (microculture): 72 hours Leukemic blood/Bone Marrow (lymphocytes) : 24 to 48 hours *Cell Division is initiated using PHA (non malignant samples) 2.Long Term Culture: For amniotic Fluid will take about 10 days to 3 weeks 12 Macroculture technique ❑ Lymphocyte separation ❑ Lymphoprep: a separation medium; a density gradient medium recommended for the isolation of mononuclear cells from peripheral blood, cord blood, and bone marrow ❑ sterile and endotoxin tested ❑ Lymphocyte-rich region 13 Culture Media ❑ Peripheral blood and BMA ▪ RPMI 1640 ▪ Fetal Bovine serum ▪ Antibiotics (Penstrep) ▪ Phytohemagglutinin (PHA) ▪ L glutamine ▪ Amniotic Fluid (same as above) plus other alternatives: ▪ Chang’s Medium D ▪ Amnio max ▪ AmnioGrow 14 Culturing Methods A. Blood Sample/Amniotic fluid cells 1. Unsynchronized 2. Synchronized ◦ Day 1- Culture Initiation ◦ Day 2- Synchronization ◦ Day 3- Synchronization Release and harvest ◦ Day 4- Slide Preparation 15 From the first subculture 30 hrs +18 hrs +7.5 hrs after Thymidine Add Add block deoxycytidine Colcemid (excess) Synchronization culture of amniotic fluid cells using excess thymidine block followed by deoxycytidine release and its application to high-resolution banding analysis of chromosomes (Hiramoto, Nahara and Kimoto, 1990) 16  17 to 18 After After 48-72 hrs after 2hrs, 45 min After 30 min hrs 0h Add Add Add Harvest Start of Fluorouracil Thymidine Colcemid cells Culture * After 45 min Ethidium bromide is added Synchronization of Lymphocyte Culture 17 Overview of the cell cycle phases and some synchronization methods. Substances that affect entry of chromosomes at junction of 2 phases are shown. * The stage of G1 phase at which lovastatin exerts its effect is not clear (Ligasova and Koberna, 2021) 18 Cell Synchronization ▪ Inhibition of DNA replication ▪ Use of inhibitors: hydroxyurea, aphidicolin, thymidine ▪ Cells are arrested at the start of S phase ▪ Release from the block; ▪ Cells continue to progress through S, G2 and M 19 2.3 Cell Harvesting 20 Arresting mitosis ❑ Use Colcemid to stop mitosis ❑ Add 2 hours before harvest (peripheral blood), ▪Add 30 minutes before harvest of Bone marrow Aspirate (BMA) culture ▪Inhibits assembly of microtubules ▪Arrests at metaphase 21 Colcemid ▪ Lyophilized (powdered form) ▪ Reconstitute in 100 ml distilled water (this packaging) ▪ 10µg/ml 22 Hypotonic solution ❑ Potassium Chloride (5.6 g dissolved in 1 liter of distilled water) ❑ KCl solution = 0.075M ❑ solution contains fewer dissolved particles than is found in normal cells and blood ❑ Prewarmed in water bath ❑ Causes cell to swell and is about to burse and chromosomes will be released to the fluid surrounding the cells ❑ Optimize the length of time cells are treated with KCl. Delicate balance. 23 2.4 Slide Preparation 24 Slide Preparation ❑ Preclean glass slides (one end is frosted) in running water for 30 minutes or so. ❑ after final washing , soak slides in 90- 95% ethanol ❑2 techniques in slide preparation ❑Air dry ❑Flame dry 25 Slide Preparation Technique Type of slide How to drop the suspension onto slide Without use of Heat Dry Slide Flat slide position With the use of Heat Wet Slide Slide at an angle position ❑ Slide preparation must take into consideration the relative humidity (RH) and temperature of the room as these affect the spreading of the chromosomes ❑ 40-45% humidity; temperature 20-22°C 26 Ageing of slides ❑ Incubation at 37°C ❑ about 2 days ❑ Complete drying of samples prior to banding 27 2.5 Staining of chromosomes 28 Before the cells are stained, the cytogeneticist must check the slides previously prepared. The level of condensation of metaphase chromosomes in most cells as well as the cell density must be considered: (1) The condensation level of the chromosomes : highly condensed (short chromosomes) or less condensed (long chromosomes). Staining time different (2) Too many cells on the slide (high cell density; overcrowding) or too few cells (low cell density). Penetration/absorption of dye is affected; overlapping of chromosomes. 29 Solid Staining of chromosomes ❑ Giemsa stain- a differential stain ❑Stains nucleus dark blue and cytoplasm to blue and pink, according to the acidity of the cytoplasm ❑Composition: azure, methylene blue, and eosin dye ❑ Specially stains the phosphate groups of DNA ❑ Attaches itself to regions of DNA that are A-T rich 30  With solid staining you can identify the different types of chromosomes based on the location of the centromere. Metaphase chromosomes 1. Metacentric 2. Submetacentric 3. Acrocentric Interphase cells Cells/chromosomes stained with Giemsa (solid staining) 31 Metaphase Chromosomes ❑ Characterized by: ▪Size and shape ▪Length (stage of mitosis when it was fixed) ❑ Location of centromere ❑ Banding pattern 32 Metaphase Chromosomes ❖ Chromosome abnormalities are best studied using metaphase chromosomes. ❖ At metaphase, chromosomes are in their maximum condensation state, thus are very visible under the microscope. 33 Groupings of Chromosomes Group Chromosome number A 1, 2, 3 Large B 4 and 5 Large submeta C 6, 7, 8, 9, 10, 11, 12 Medium meta D 13, 14, and 15 Large acrocentric E 16, 17, and 18 Small submeta F 19, and 20 Small meta G 21, 22 Small acro The average human chromosome has just under 5 cm of DNA 34 Measurement features of chromosomes ❑ Centromeric index (CI) – the ratio of the length of the short arm of the chromosome to that of the total chromosome; expressed as % CI = [p/(p+q)x100] ❑ Arm ratio (AR)=Length of long arm/length of short arm AR = q/p 35  Measurement features of chromosomes ▪ Relative length of each chromosome to the total length of a haploid set ▪ The relative position of the centromere is constant, which means that that the ratio of the lengths of the two arms is constant for each chromosome. 36 Types of chromosomes (Location of centromere) http://www.expertsmind.com/ 37  Centromere Chromosome Size Chromosome position (Mbp) (Mbp) 1 125.0 metacentric 247.2 2 93.3 submetacentric 242.8 3 91.0 metacentric 199.4 4 50.4 submetacentric 191.3 5 48.4 submetacentric 180.8 6 61.0 submetacentric 170.9 7 59.9 submetacentric 158.8 8 45.6 submetacentric 146.3 9 49.0 submetacentric 140.4 10 40.2 submetacentric 135.4 11 53.7 submetacentric 134.5 12 35.8 submetacentric 132.3 13 17.9 acrocentric 114.1 14 17.6 acrocentric 106.3 15 19.0 acrocentric 100.3 16 36.6 metacentric 88.8 17 24.0 submetacentric 78.7 18 17.2 submetacentric 76.1 19 26.5 metacentric 63.8 20 27.5 metacentric 62.4 21 13.2 acrocentric 46.9 22 14.7 acrocentric 49.5 X 60.6 submetacentric 154.9 Y 12.5 acrocentric 57.7 38 STAINING METHODS USED IN METAPHASE CHROMOSOMES 1.Differential Staining 2.Selective Staining techniques 39 A. Classical Differential Staining Methods ❑GTG Banding ❑Q Banding ❑R-banding 40 Banding patterns ❑ Permit structural definition and differentiation of chromosomes ❑ Alternate dark and light bands/Bright and dark ❑ Bands reflect different base composition ❑ C-G rich band or A-T rich band 41 G-bands by Trypsin Using Giemsa (GTG banding) ❑ Giemsa staining solution 5% ❑ Chromosomes (nucleic acid) are treated with Trypsin prior to Giemsa staining. ❑ Trypsin is a proteolytic enzyme ❑ Produces Dark and Light bands ❑ Comings 1978 42 G-Banded metaphase chromosomes arrested at various times of metaphase (early to late) 43 Q-bands by Fluorescense Using Quinacrine (QFQ or Q-banding) ▪ Staining solution : Quinacrine (0.5 %) in Sorensen buffer, pH=7.0 ▪ Light sensitive stain (wrap bottle in foil) ▪ Needs a Fluorescence Microscope ▪ Needs UV Optics with appropriate filters (excitation filter 450-490 nm) ▪ Casperson et al., 1971 44 https://www.chegg.com/learn/biology/introduction-to-biology/banding-pattern 45  A B In figures A and B, the Y chromosome (arrows) shows remarkably bright fluorescence and is the most intense in the entire complement. Mayo.edu 46 Comparing G and Q Bands Dark bands/Bright Bands Pale Bright/Dark bands A-T Rich region of G-C rich region of chromosomes chromosomes Gene poor Gene rich Condense early Condense late Replicate late Replicate early Positive bands Negative bands 47 R- Bands by Heat Using Giemsa (R-banding) ❑ Reverse banding ❑ Treatment of slides at high temperatures with various buffers; then ❑ Giemsa staining ❑ Mechanism not fully understood ❑ Heat denatures chromosomal proteins (histones) as well as the A-T rich regions of the DNA ❑ G-C rich regions are left in a native configuration ❑ Summer, 1982 48 R (Reverse) bands 49  G-banded and R-Banded Metaphase Chromosomes http://vignette2.wikia.nocookie.net/ http://image.slidesharecdn.com/ 50  Banding Treatment Bands Method G-banding Chromosomes are subjected Dark and light bands to trypsin digestion Q banding Chromosomes are stained Bright and dark bands with fluorescent dye quinacrine mustard R banding Chromosomes are heat- Light and Dark bands denatured in saline before stained with Giemsa; Essentially reverse of G- banding 51 Selective BANDING METHODS ❑ C-Banding (centromere) ❑ T-Banding (telomere) ❑ Ag-NOR banding 52 Forms of chromatin (1) euchromatin contains actively expressed chromosomal regions (genes) (2) heterochromatin gene loci within these regions are silenced 53 C-banding ▪ Stains the Centromere; ▪ Stains the Constitutive heterochromatin domains found throughout the chromosomes of eukaryotes. ▪Mostly found at the pericentromeric regions of chromosomes and also in telomeres ❖ Constitutive heterochromatin found on chromosomes 1, 9, 16, 19 and Y. 54 C – Banding ❖ Constitutive heterochromatin account for about 200Mb or 6.5% of the total human genome; ❖ mainly made up of high copy number repeats: satellite repeats, minisatellites, microsatellites ❖ their repeat composition makes them difficult to sequence, so only small regions have been sequenced 55 C-banding staining http://web.udl.es/usuaris 56 Chromosome heterochromatin variants 57 Suggested Reading Vorsanova SG et al. 2007. Variability in the heterochromatin regions of the chromosomes and chromosomal anomalies in children with autism: identification of genetic markers of autistic spectrum disorders. Neurosci Behav Physiol. 2007 Jul;37(6):553-8. 58 T-banding ▪ T-banding involves the staining of telomeric regions of chromosomes using either Giemsa or acridine orange after controlled thermal denaturation. ▪ T bands apparently represent a subset of the R bands because they are smaller that the corresponding R bands and are more strictly telomeric. 59 Staining the telomeres 60 Advanced Banding Methods 1. High resolution banding 2. Sister Chromatid Exchanges 3. Restriction Enzyme Digestion 4. NOR (Nucleolar Organizing region) or AgNOR Silver Staining 61 Sister chromatid exchange (SCE) ❑A process that occurs during DNA replication ❑An exchange of genetic material between parental strands in the duplicated chromosomes (break and rejoin). ❑Considered to be conservative and error-free ❑No information is altered during reciprocal interchange by homologous recombination. 62 SISTER CHROMATID EXCHANGES ▪ It was first discovered by using the Giemsa staining method on one chromatid belonging to the sister chromatid complex before anaphase in mitosis ▪ sister chromatid exchange (SCE) test is a relatively simple assay, sensitive to most mutagenic compounds 63 SCE ▪ Can be visualized using Giemsa/bromodeoxyuridine (BrdU) BrdU is a Thymidine analog which is introduced in the desired chromatid ▪ BrdU is incorporated into replicating DNA for 2 successive cell cycles and subsequently subjected to photodegradation. ▪ Result: sister chromatid differentiation ▪ (1) a faintly stained chromatid containing bifilarly (two threads) BrdU substituted DNA ▪ (2) a brightly stained sister-chromatid containing unifilarly(one thread) BrdU substituted DNA 64 Sister-chromatid Exchanges BrdU Colcemid 0h 70h 24 h 48 h 72 h M1 M2 https://we.vub.ac.be/~cege/volders/ENG/tests/SCE.htm 65 Diagrammatic illustration of mechanism of sister chromatid exchanges. Solid bars indicate the native DNA strand, while hatched bars indicate BrdU incorporated strand (Verma and Babu, 1995) 66 Sister chromatid exchange (SCE) 67 SCE Mechanism ▪ Reason for SCE not known ▪ Seen in most normal Eukaryotic cells (human cells) ▪ Frequency of 2-20 with mean frequency of 5-8 ▪ Maximum frequency of SCE is observed between ages 30-40 years 68 SCEs ▪ Four to five sister chromatid exchange per chromosome pair, per mitosis is in the normal distribution. ▪ 14-100 exchanges is not normal and presents a danger to the organism. ▪ Bloom syndrome patients have recombination rates more than 10 to 100 X above normal. ▪ Frequency of SCE may be also related to tumour formation 69 Bloom syndrome ❑ An inherited disorder (autosomal recessive) characterized: ▪ by short stature, growth retardation ▪ a skin (butterfly) rash that develops after exposure to the sun, ▪ a greatly increased risk of cancer ▪ compromised immune system ▪ a mutation (change) in the BLM gene that causes cells to have abnormal breaks in the chromosomes https://slideplayer.com/slide/13997357/ 70 Molecular Mechanism of SCE Wilson and Thomson 2007 71 Ag-NOR (Silver) Staining Staining of Satellites in acrocentric chromosomes Secondary constriction (satellite stalk) of acrocentric chromosome carries the genes for NOR http://jvi.asm.org/conten 72 Silver Staining (AgNO3) Representative nucleolar organizing region-banded partial metaphases illustrating double satellites (A) and an isodicentric chromosome with satellites at both ends (B) 73 AG Staining: Satellites and stalks (only on acrocentric chromosomes) 74 Fragile X syndrome ❑ Can be diagnosed by cytogenetics ❑ Caused a change to a gene on the X-chromosome called the FMR1 gene. ❑ Gene encodes a protein (FMRP) is needed for normal brain development ❑ This syndrome is caused by a mutation in this gene (FMR1 gene); ❑ expansion of the CGG trinucleotide repeat in the 5’ untranslated region of the gene ❑ No cure for FXS, treatment to help learn important skills 75 Cell cultures that do not yield mitotic cells (metaphase) cannot be processed for karyotyping. 76 3. Chromosome Analysis 77  Karyotyping ❑ the process of pairing and ordering all the chromosomes of an organism (e.g. human) ❑ providing a genome-wide snapshot of a person’s chromosomes 78 Terms to Use ❑ Karyogram – a systematized array of chromosomes prepared either by drawing, digitized imaging or by photographs. ❑ Karyotype is the use of nomenclature to describe the normal or abnormal; constitutional or acquired chromosome complement of an individual. ▪ 46,XX or 46,XY normal karyotypes ▪ 47,XX,+21 female with three copies of chromosome 21 ❑ Based on the ISCN International System of Human Cytogenetic Nomenclature ❑ Ideogram is a graphical representation of chromosomes 79 A normal male karyogram. Chromosomes are long (less compact state) so more bands are seen. 80 A normal female karyogram. Chromosomes are short (condensed state). 81 In today’s technology, an advanced microscope can be attached to a computer (Imaging system) where softwares are installed to execute automated karyotyping. But the program is not perfect, some errors in chromosome arrangement still happen so the cytogeneticist must correct this errors by moving the chromosomes to their correct position in the template provided. 82 The Imaging system has a software (e.g. Applied Spectral Imaging) that provides an ideogram for each banded chromosome as a guide in the correct positioning of the chromosomes. 83 An ideogram of human of chromosomes. Regions in red represent centromeres, stripe red are the heterochromatin regions. Dark and light bands correspond to the banding pattern produced by G-banding. 84 Hctcíockíomatic ícgio⭲s (CG-CNVs) a⭲d c"ckíomatic :aíia⭲ts (EVs) or tkc k"ma⭲ gc⭲omc aíc kigkligktcd i⭲tkis sckcmatic dcpictio⭲ or a kaploid sct or k"ma⭲ ckíomosomcs (ícscaíckgatc.⭲ct) 85 Chromosome can be identified using three important features: centromere position (slide # 37), size (see slide # 38) and banding pattern (slide # 80). 86  ncbi.nlm.nih.gov www.ncbi.nlm.nih.go 87 What is the role of cytogenetics? ▪Key role in the detection of chromosomal abnormalities associated with cancer (malignancies) ▪Helps in the diagnosis and classification of diseases ▪Characterization of new alterations/mutations ▪Guide clinicians in planning treatment regimen ▪Monitoring the status of the disease 88 Research Applications of Cytogenetics 89 Bleomycin Sulfate (Blenoxane) ▪ An anti-cancer drug ▪ from Streptomyces verticullus ▪ A radiomimetic agent: mimics the action of ionizing radiation) ▪ Direct and indirect effects to the DNA molecule 90 Scoring of chromatid breaks (ctbs) in Bleomycin Assay Mean break per cell b/c = ratio between total number of chromatid breaks scored and total number of cells screened or analyzed x 100% < 0.80 b/c = hyposensitive to the action of bleomycin 0.80 – 1.0 b/c = borderline of sensitivity > 1.0 b/c = hypersensitive to the action of bleomycin 91  Normal metaphase spread stained with Giemsa Patient with numerous chromatid breaks Bleomycin Assay/ Chromosome Fragility Assay 92 Enriquez MD, Santos DR, Postor IQ, Cheng KR, Hamoy GL, Carag AR, Buhain DV, Mapua C, and Natividad FF. 2008. Evaluating Cancer Susceptibility of Jeepney Drivers in Metro Manila Using the Chromosome Fragility Assay. St. Luke’s Journal of Medicine. January-June Vol. 4 No.1: 69-73 93 Chromosome Fragile Sites (CFS) ❑ Regions of chromosome instability that show gaps or breaks when cells are exposed to particular culture conditions (e.g. bleomycin treatment) ❑ Related to Cancer susceptibility ❑ Breakpoints in recurrent chromosome mutations ❑ Sites of viral integration 94 FRAGILE SITES in CHROMOSOMES: possible model for the study of spontaneous chromosome breakage. 95 Mutagen sensitivity is an indirect indicator of DNA repair competence. Sensitivity ( 1.0 > b/c) to the mutagen (bleomycin) may be taken as high risk to develop environmentally-induced cancer 96 BLM Test (Hsu et al., 1989) ❑ Peripheral blood is cultured following the conventional protocol. Lymphocytes are cultured for 72 hours. Five hours before harvest (67h), bleomycin is added to the cultures (30 µg/ml); colcemid treatment is added on the 70h. ❑ Chromosomes are harvested, slides are prepared and chromosomes are stained with Giemsa (solid staining) ❑ Fifty to 100 metaphase are scored from each subject in the study. ❑ Chromosomes are analyzed for the presence of chromatid gap, break, chromosome gap, chromosome break 97 Cytogenetics in Medicine 98  Fragile X syndrome ❑Can be diagnosed by cytogenetics ❑Caused by a change/mutation to a gene on the X- chromosome called the FMR1 gene. ❑ Gene encodes a protein (FMRP); needed for normal brain development ❑cause by expansion of the CGG trinucleotide repeat in the 5’ untranslated region of the gene ❑No cure for FXS, treatment to help learn important skills 99 Fragile X Syndrome More males are severely affected by fragile X syndrome than females EurekaAlert/medicalnewstoday.com 100 Fragile X syndrome is caused by the expansion of CGG tri-nucleotide repeats on the X chromosome at the FMR-1 gene locus (Xq27.3). 5-fluoro-2'-deoxyuridine (FUdR), an inhibitor of thymidylate synthetase is added to cultures to be able to see the expression of this fragile site. 101 Hematologic malignancies/Leukemia ❑ Cytogenetic analysis is the most important diagnostic tool for determining prognosis in hematologic malignancies like acute and chronic leukemia. ❑ Most leukemia are characterized by clonal reciprocal translocation (exchange segments between 2 chromosomes) ❑ Hall mark of chronic myelogenous leukemia (CML) is the reciprocal translocation between chromosomes 9 and 22. ❑ Such exchange of segments result to the fusion of two genes: ABL on chromosome 9 and BCR gene on chromosome 22 ❑ BCR-ABL fusion (pathogenic fusion) 102 Chromosomes – Genes BCR Ph ABL 103  Abnormal Gene Mutation Chromosomes Not all Gene Mutations can be seen/observed at the level of the chromosomes 104 Summary of Steps 105  Methodology of Chromosomal Analysis RPMI 1640 on the 70th hour add colcemid Add 0.075 M KCl L-Glutamine incubate at 37 oC Antibiotics for 22 mins. Fetal Bovine Serum Phytohemagglutinin On the 72nd hour,centrifuge CULTURE SET-UP at 1000 rpm for 10 mins. HARVEST Analysis of Metaphase Spreads Isolation of LYMPHOCYTES Splash 2 separate drops Centrifuge 4X at 1100 rpm, 106 suspend cells in Carnoy’s Fixer (Methanol + Acetic Acid) R and B, SLMC Molcyto lab 107 Cytogenetics Workstation 108 Imaging ▪ The science and technology of acquiring spatial and temporal data information from objects for the purpose of obtaining information. ▪ Digital imaging is the most advanced and applicable method where data are recorded using a digital camera, such as a charged Coupled Device (CCD) 109  Abnormal Karyotypes in two Filipino colon cancer patients A complex karyotype of CCA Karyogram of patient CCA 318. Note numerical and 321 showing trisomy 7 structural chromosome abnormalities. COLON CANCER HAS MANY POSSIBLE KARYOTYPES 110 Cytogenetic techniques are central to the assignment and localization of genes to chromosomes and thus to the construction of genetic maps. 111 Additional Readings Ligasová, Anna, and Karel Koberna. 2021. "Strengths and Weaknesses of Cell Synchronization Protocols Based on Inhibition of DNA Synthesis" International Journal of Molecular Sciences 22, no. 19: 10759. https://doi.oíg/10.3390/ijms221910759 Zbigniew Darzynkiewicz, H. Dorota Halicka, and Hong Zhao. 2011. Cell synchronization by inhibitors of DNA replication induces replication stress and DNA damage response: analysis by flow cytometry. 2012. Methods Mol Biol. 761: 85–96. doi:10.1007/978-1-61779-182-6_6 112 Thank you for your attention! 113