Cytogenetic Techniques: Semester 1 FINALS 2024-2025 PDF

CYTOGENETIC TECHNIQUES Semester 1 FINALS | A.Y. 2024-2025 transcriptionally inactive areas OUTLINE of the genome. I. KARYOTYPING II. BANDING PATTERN III....

CYTOGENETIC TECHNIQUES Semester 1 FINALS | A.Y. 2024-2025 transcriptionally inactive areas OUTLINE of the genome. I. KARYOTYPING II. BANDING PATTERN III. FLUORESCENT IN-SITU HYBRIDIZATION CONVENTIONAL KARYOTYPE (FISH) PREPARATION IV. COMPARATIVE GENOME HYBRIDIZATION V. SPECTRAL KARYOTYPING VI. POLYMERASE CHAIN REACTION 1. Cells are arrested at the metaphase staged by colcemid. KARYOTYPING 2. Cells are fixed and placed on a slide 3. Proteolytic treatment (Trypsin) ➔ The accurate organization (matching and alignment) of the chromosomal content of 4. Staining with Giemsa any given cell type ➔ Chromosomes are arranged and ➔ Giemsa dye generates distinct numbered by size (largest to smallest). chromosome-specific patterns called G-bands Cancer ➔ COLCEMID - mitotic inhibitor that disrupts the formation of mitotic spindles, ➔ Acquisition of chromosome aberrations is effectively blocking cells at metaphase the main mechanism by which cancer cells evolve. ➔ G-banding has been divided into regions, ➔ Chromosome changes can lead to bands, and subbands. abnormal expression of many genes. METHODS TO PREPARE THE KARYOTYPE BANDING PATTERNS ➔ The most common karyotype preparation ➔ Banding patterns can distinguish method used for years is the conventional chromosomal abnormalities or structural or classic cytogenetic technique rearrangements such as translocations, ➔ Cells are cultured, metaphases are deletions, insertions and inversions. obtained, and the chromosomes with an appropriate stain are studied Burkitt’s Lymphoma CONVENTIONAL CYTOGENETICS ➔ Translocation of chromosome 8 and ➔ Used to determine the numerical chromosome 14 - most common cause of chromosomal abnormalities or structural burkitt's lymphoma rearrangements. ➔ MYC gene on chromosome 8 ➔ Examples: ○ Gains or losses of segments ○ Rearrangements within and ★ Harsher treatment of chromosome (87C between chromosomes for 10 min) before Giemsa staining can produce a pattern called R banding Euchromatin Transcriptionally active (opposite to the G banding pattern) areas of the genome in ★ R banding - can stain the euchromatin which histone association region with the DNA has been ★ The centromere can also be seen by reduced. staining (Alkali treatment of the chromosome) Hetero- Tight association of histones and ★ Referred to as C bands chromatin ★ Centromere region - rich in DNA, and represents the heterochromatin and is resistant to Trypsin NOT FOR SALE | AG ♡ LECTURE 1 | CYTOGENETICS – Cytogenetic Techniques NOT FOR SALE | AG ♡ ➔ Can be performed in metaphase preparations or interphase cells. G - band Heterochromatin (Trypsin treatment) R - band Euchromatin (87C for 10 minutes) METAPHASE FISH ➔ Preparation of metaphase FISH samples begins with the culture of cells for 72 C - band Heterochromatin/Centromere hours. (Alkali treatment) ➔ 4 minutes before harvesting, colcemid is added to the culture. KARYOTYPING PROTOCOL ➔ The cells are then suspended in a hypotonic media and then fixed with ➔ Marrow blood > Cell culture > Metaphase methanol/acetic acid. arrest w/ colcemid > Hypotonic treatment > Fixation > Spreading > Banding > Karyotyping INTERPHASE FISH ○ Hypotonic Treatment - to lyse the cell, exposing the nucleus and the ➔ Cell culture is not required in Interphase FISH chromosomes ○ Carnoy’s fixative (fixation step)- ➔ Used for samples that do not divide well Methanol and glacial acetic acid in culture ➔ Prenatal samples, tumors and FLUORESCENCE-IN-SITU HYBRIDIZATION hematologic malignancies ➔ Karyotyping disadvantage: Limited 1. Optimal results are obtained if resolution fresh cells are incubated overnight after deposition on the slide ➔ In situ hybridization (ISH) technology provided another dimension to the study 2. After aging overnight, cells are of chromosomes, facilitating the treated with protease visualization of DNA sequences on 3. Dehydrated using ethanol chromosomes and in cells at a molecular 4. Fixed with 1% formaldehyde level 5. Addition of fluorescent probe ➔ ISH technique involves the use of a 6. Visualization under fluorescent nonradioactive probe (such as biotin) for microscope indirect labeling through nick translation ➔ The hybridization between DNA probe and target sequence could be visualized FISH PROBES through avidin or streptavidin fluorescent labeling ➔ Three broad types of FISH probes are used in clinical genetics laboratories, each with different applications STANDARD FISH PROTOCOL: ○ WCP Probes - mark entire chromosome of interest 1. Sample pretreatment 2. Denaturation of probe and sample ○ Centromeric Enumeration Probes - 3. Hybridization of probe to target cells hybridize to centromeric regions of (annealing) each chromosome 4. Post hybridization washing 5. Detection (epifluorescence microscope ○ Locus-Specific Identifier Probes - specific regions/locus of ➔ Advantage: Increased resolution, chromosomes applicable for clinical diagnosis and research ➔ FISH is based on fluorescently labeled probes that hybridize to unique DNA sequences along the chromosomes. NOT FOR SALE | AG ♡ LECTURE 1 | CYTOGENETICS – Cytogenetic Techniques NOT FOR SALE | AG ♡ ADVANCES IN FISH TECHNOLOGY 1. TWO oligonucleotide primers 2. DNA polymerase (taq polymerase) ➔ Numerous methodological advances in molecular cytogenetics technology have 3. dNTPs (deoxyNucleosideTriphosphate) been developed: ○ Comparative Genome Hybridization (CGH) ➔ Primers will bind to a region of the DNA to initiate the replication of DNA ○ Spectral Karyotyping (SKY) ➔ Oligonucleotides – acts as primers for the ○ Multicolor FISH (MFISH) DNA synthesis reaction. They define the amplified region. COMPARATIVE GENOME HYBRIDIZATION ➔ DNA polymerase will catalyze the synthesis of DNA and will produce the (CGH) newly synthesized strand in a 5’ to 3’ direction ➔ Major advantage: only the DNA from the tumor cells is needed for analysis, does ➔ DNA polymerase can extend the annealed not require metaphase chromosomes primers by adding on dNTP (deoxyribonucleoside Triphosphate) ➔ Used for genome-wide screening for chromosomal copy number changes ➔ DNA polymerase can extend the annealed (gains/duplications/losses/deletions) primers by adding on dNTP (deoxyribonucleoside Triphosphate) ➔ Introduced to study chromosomal abnormalities that occur in solid tumors ➔ deoxyNucleosidesPhosphate (dNTP) – and other malignancies. required for the synthesis of complementary DNA strands. ➔ DNA polymerase (taq polymerase) – thermostable DNA polymerase I enzyme from Thermus aquaticus. ○ The thermostability of Taq polymerase is an essential requirement in PCR. MFISH AND SKY Yellowstone National Park ➔ Uses specific probes each with varying ➔ The bacteria Thermus aquaticus causes amounts of dyes. the bright coloration of the hot springs located in Yellowstone National Park ➔ Different pairs of chromosomes show unique spectral characteristics. ➔ Thermus aquaticus: ➔ DIFFERENCE: the way they catch the ○ Rod-shaped, non-motile, colored images. pigmented ➔ MFISH - fluorescent filters ○ Gram stain: Negative ➔ SKY - spectroscopy ○ Metabolism: Obligate aerobe ○ Growth media: Castenhols TYE POLYMERASE CHAIN REACTION (PCR) ○ Growth temperature: 70C ➔ In vitro enzymatic reaction to amplify a ➔ The PCR reaction will go through different defined DNA region temperature stages that may denature or ➔ End-point assay to examine the existence disable the non thermostable polymerase. of target DNA in a sample ➔ A PCR reaction normally uses the ff: NOT FOR SALE | AG ♡ LECTURE 1 | CYTOGENETICS – Cytogenetic Techniques NOT FOR SALE | AG ♡ Three steps of a PCR reaction ○ 3’ END TERMINATES BECAUSE A NEW CYCLE WILL START ➔ DENATURATION ➔ Repeated cycle of Denaturation-Annealing-Extension ○ In a typical PCR experiment, the target DNA is mixed with Taq ➔ In subsequent cycles, the number of Polymerase, the two correct amplification products oligonucleotide primers, and a accumulates in an exponential fashion supply of dNTPs. (doubling during each cycle) ○ The reaction is started by heating ➔ After 30-40 cycles → 1 billion products the mixture to 94C derived from each starting molecule ○ Hydrogen bonds that hold the two polynucleotides of the double helix Amplicons refers to the product of are broken amplification reactions ○ The target DNA becomes denatured into single stranded Three steps of a PCR reaction molecules Analysis of PCR products: ➔ ANNEALING ➔ Conventional PCR ○ The temperature is then decreased to 50-60C ○ To evaluate whether a PCR reaction is successful, one has to ○ This allows the primers to attach examine the PCR products. to their complementary sequences ○ Simplest and easiest procedure is ○ TWO oligonucleotide PRIMERS to analyze the PCR products by agarose gel electrophoresis. ➔ EXTENSION ➔ (+) Expected Band – PCR reaction ○ DNA synthesis can now begin successful when the temperature is raised to 72-74C ➔ (-) Expected Band / Multiple Bands – something has gone wrong, and the ○ Taq polymerase experiment must be repeated. DENATURATION 94 °C Breaking Hydrogen bonds ANNEALING 50-60C Two Oligonucleotides (Primers) EXTENSION 72-74C Taq Polymerase ➔ In the first cycle of PCR, a set of “long products” is synthesized ➔ Identical 5’ ends but random 3’ ends → Terminates by chance ➔ In the first cycle of PCR, a set of “long products” is synthesized ➔ Identical 5’ ends but random 3’ ends → Terminates by chance NOT FOR SALE | AG ♡