UNIT 6 and 7 Molecular Cytogenetics Techniques PDF

CYTOGENETICS College of Allied Health Sciences Bachelor of Science in Medical Laboratory Science 1st Semester | A.Y. 2024-2025 Unit 6 and 7: Techniques in Molecular Cytogenetics Outline: ◼ Karyotyping is a test to examine 1.1 Fluorescence In Situ...

CYTOGENETICS College of Allied Health Sciences Bachelor of Science in Medical Laboratory Science 1st Semester | A.Y. 2024-2025 Unit 6 and 7: Techniques in Molecular Cytogenetics Outline: ◼ Karyotyping is a test to examine 1.1 Fluorescence In Situ Hybridization chromosomes in a sample of cells. This test 1.2 Comparative Genomic Hybridization can help identify genetic problems as the 1.3 Spectral Karyotyping cause of a disorder or disease How To Do Karyotyping? Molecular Cytogenetic Techniques - FISH: a powerful tool for identifying the presence of specific genes or chromosomes within the cell by attaching fluorescent tags to DNA sequences. This is also useful in detecting abnormalities or gene - We need to culture cell samples rearrangements. and add chemical agents to - CGH: This method compares the our culture medium to arrest the DNA sample to normal control, cells at their metaphase stage. identifying variations in DNA Then, we have to incubate our copy number, like gains and cultured medium. After losses of chromosomal regions. incubation, we need to harvest - SKY: This technique allows the cell sample, fix it on the visualization of all chromosomes slide, and stain it. After staining, in different colors by using a full we need to conduct spectrum of fluorescence it visualization and helps identify complex rearrangements of chromosomal rearrangements chromosomes. such as translocations and inversions. Chromosomal Banding Techniques KARYOTYPING - Karyotyping and Chromosomal Banding are classic cytogenetics. - Karyotyping is used to visualize and analyze the complete set of chromosomes. ◼ The chromosomes are depicted (by rearranging a photomicrograph) in a standard format known as a karyogram or idiogram. MARIANO | BSMLS – 2A 1 CYTOGENETICS College of Allied Health Sciences Bachelor of Science in Medical Laboratory Science 1st Semester | A.Y. 2024-2025 ◼ (1991)-The first application of FISH to plant FLUORESCENCE IN-SITU HYBRIDIZATION (FISH) cytogenetics was the work of Leitch et al. (1991) Fluorescence in-situ hybridization is a molecular cytogenetic technique that tags Principles of Fluorescence in situ Hybridization genetic material with fluorescent molecules. It is a technique for mapping the The basic location of genes onto chromosomes. elements of FISH are a DNA probe and a target sequence. Before hybridization, the DNA probe is labelled by various means, such as nick translation, random primed labelling, and PCR. The labelled probe and the target DNA are denatured. - FISH can be used to detect Combining the denatured probe and small deletions and duplications target. that are not visible in microscope analysis. - HYBRIDIZATION: Fish relies on the - Uses a very small chemical that principle of hybridization where glows brightly when it detects a single-stranded DNA probe the specific region in a binds to its complementary chromosome. sequence in the target DNA. - SPECIFICITY: The probe is designed to bind only to its History specific target sequence ensuring high specificity in The technique detecting the target DNA. was first - FLUORESCENCE: The probe is developed in labeled with a fluorescent 1982. But its molecule or we call it use and fluorophore allowing application came more visualization of the target into play after sequence under a fluorescence 1990 because microscope. by then easily accessed probe generation and detection methods and many other techniques were introduced. Indications for FISH History and Development Diagnosis 1. Culture not successful ◼ (1969)- In situ hybridization - For example, when the technique was traditional karyotyping fails. developed by Traditional karyotyping failure Joseph G Gal and Mary Lou requires growing cells in the Pardue and culture so in the event that the John et culture was not successful, we al.(1969) could perform FISH. ◼ (1985)- The non isotopic in situ hybridization using biotin labeled DNA probes was first introduced in plant species by Rayburn and 2. Expected abnormality not Gill seen MARIANO | BSMLS – 2A 2 CYTOGENETICS College of Allied Health Sciences Bachelor of Science in Medical Laboratory Science 1st Semester | A.Y. 2024-2025 3. To confirm a suspected abnormality - The sequence of the probe is 4. To detect a cryptic complementary to the translocation To monitor response to treatment sequence of interest in the Engraftment in sex – mismatched BM target DNA, meaning the transplant nucleotides of the probe will pair up with the opposite bases - In cases of bone marrow on the target sequence. Probe transplant, FISH can be used to is like a search tool that monitor the engraftment of attaches to a matching DNA donor cells especially in sex- sequence in a sample allowing mismatched transplants where Y scientists to detect or study the chromosome specific probes part of your DNA. can be used. - The length of the probe depends on the sequences that were going to be detected. Sample Type - If we are going to detect smaller sequences, we use shorter ◼ 1. Frozen sections probes. - If we are going to detect larger - Tissues that have been cryo- chromosomal regions, we use preserved. longer probes. - Probes can be labeled in different ways to allow ◼ 2. Paraffin-embedded sections detection. Labelling is a crucial step coz it helps in detecting - Commonly used in physiological DNA sequences when target or pathology laboratories where samples are hybridized. tissues are preserved in a - Primary methods for probe paraffin wax. Special critic main labeling; steps are required to remove Indirect: incorporation the paraffin and digest the of hapten (fluorescent tissue to allow probe tag but they are penetration. essential for increasing sensitivity and amplifying the ◼ 3. Cells in suspension detection signal (e.g. biotin and digoxigenin) - Examples are blood, bone to the DNA. It also marrow, amnio-sites and salines. requires secondary It requires specific processing detection using steps to ensure proper cell fluorescent -labeled morphology and chromosome antibody spreading. (fluorochromes, rhodamine, Texas Red, fluorescein) What are Probes? Direct: do not require ◼ Probe is a synthesized fragment of DNA or secondary detection RNA of variable length which can be because the radioactively labeled fluorochrome directly ◼ The size maybe varies from 100-1000 bases attached to the probe long. nucleotides. Since it is ◼ The probe thereby hybridizes to single- directly attached, there stranded nucleic acid (DNA or RNA) whose is a direct visualization base sequence allows probe-target base after the fluorescent pairing due to complementarity between excitation. the probe and target. MARIANO | BSMLS – 2A 3 CYTOGENETICS College of Allied Health Sciences Bachelor of Science in Medical Laboratory Science 1st Semester | A.Y. 2024-2025 ◼ the probe is tagged (or "labeled") with 2. Whole chromosome a molecular marker of either radioactive or fluorescent molecules. - Cover the entire length of a - Specific tags thar are commonly chromosome. used with these probes - Used to visualize an entire - TAGS w/c are also as markers or chromosome making it easy to labels or molecules attached to detect large structural changes the probes to help us where like translocations or complex they bind. In both direct and rearrangements. indirect, these tags are crucial - Example: a probe for the whole for visualizing the probe’s chromosome, can help detect hybridization to specific involvement in a translocation sequences of DNA. with another chromosome. - Most commonly used tags for FISH: (1) fluorescent molecule (modern probes are labeled 3. Unique sequence with fluorescent dyes). When these probes hybridize with the - Probes bind to specific genes or target DNA, the bound probes unique DNA sequences in the emit light under a fluorescence genome. microscope allowing for - Used to detect specific genes or visualization. (either direct and unique chromosomal loci. indirect) (2) Haptens which we - Example: detection of BCR- use in indirect labeling. (Biotin - ABLG fusion in the chronic detected using streptavidin myeloid leukemia. which binds tightly to biotin and is linked to a fluorescent dye or enzyme for signal detection. Digoxigenin – detected using anti-digoxigenin antibodies w/c Types of Probes are attached to fluorescent dye/fluorochrome/enzyme to - Refer to more specific produce visible signals. classifications within each category that is often based to probe’s structure, design or Categories of Probes application. - Suit in particular experimental - Refer to broad classification based on needs or analytical methods the function or genomic target of the probe. It describes the kind of information the probe provides and what part of the genome it is designed 1) Locus-specific probes – a type of unique to bind. sequence probe; used to determine the presence or absence of the 1. Repetitive sequence rearrangements of specific genes making them highly useful in diagnosing genetic disorders, and cancer and identifying - Probes w/c target repetitive specific gene mutations. DNA sequences that are found throughout the genome. (ex: 2) Alphoid or centromeric repeat probes – centromere and telomere a type of repetitive sequence probe; contain highly repetitive DNA primarily used to detect aneuploidy by identifying the presence and absence of sequences. specific chromosomes. A probe for the - Use in chromosome centromere of chromosome 21 can be used enumeration and to identify to detect Trisomy 21. specific chromosomal regions. 3) Telomere probes – a repetitive sequence probe; used to study the integrity of MARIANO | BSMLS – 2A 4 CYTOGENETICS College of Allied Health Sciences Bachelor of Science in Medical Laboratory Science 1st Semester | A.Y. 2024-2025 telomeres and to detect structural changes involving the end of your chromosome like translocation. (ex: telomere shortening in - Identify first the DNA sequence diseases like Dyskeratosis Congenita) that we want to visualize and then after that we prepare our 4) Whole chromosome probes – under whole samples by fixing 5% chromosome probe/chromosome paraformaldehyde to preserve libraries/chromosome paints; paint the their structure and DNA integrity. entire chromosome making them useful for - DENATURATION: heating the identifying structural abnormalities like double-stranded DNA in the translocations, large deletions, and sample so that two strands will duplications in chromosomes. separate becoming a single- stranded DNA. It is crucial for allowing the probe to bind to its complementary DNA sequence during hybridization. - The sample is often heated in a controlled environment which disrupts the hydrogen bonds between the DNA strands that’s why it is converted to single- stranded DNA. ◼ Step II – Hybridization Types of FISH Application of probe DNA to slide & overnight incubation at 37°C Binding of probe DNA to target DNA. - The labeled single-stranded DNA probe is applied to the prepared sample often containing cells or chromosomes. - Occurs moist-warmed Process of Fluorescent In Situ Hybridization environment ensuring the probe can be able to bind to its target ◼ Step I – Denaturation sequence. Conversion of - Usually the cells are incubated double- under specific conditions to stranded DNA allow the probe to hybridize to into single- its complementary target stranded DNA. sequence in the DNA (usually incubate at 37 degrees Celsius and incubate overnight) ◼ Step III – Post-hybridization washing & detection Washing of unbound probe DNA. Application of counter stain MARIANO | BSMLS – 2A 5 CYTOGENETICS College of Allied Health Sciences Bachelor of Science in Medical Laboratory Science 1st Semester | A.Y. 2024-2025 - Observing the hybridized sample under a fluorescence microscope. The fluorescent- labeled probe will emit light - The main goal is to remove the when exposed to appropriate unbound or non-specifically wavelengths allowing scientists bound probes. The cells are to see where the probe has washed using a series of washes bound to the target DNA. with increasing stringency. This is - Patterns of fluorescence will be to ensure only probes bound to observed and then as well as the target DNA remain. abnormalities will be analyzed - After the washing technique, based on the signals emitted by we can apply counter stain and the fluorescence probes. after that detection na. - Target sample: the biological material such as our cells, tissue ◼ Step IV – counter stain Application of sections, and body fluids from counter stain. which we are studying or analyzing the DNA. In short words, the target sample contains or source of the target - DAPI (4’6 – diamidino – 2 – DNA. phenylindole) will bind to the - Target DNA: refers to the specific DNA and stains the entire sequence of DNA w/in the chromosome providing of visual sample that a probe is designed context for fluorescent signal to locate and bind to during a from the probe. So, basically, molecular hybridization the counter stain will make it experiment. It is the portion of easier for us to visualize the DNA that we are interested to overall structure of the study or identify. Target DNA is chromosome under the present in the cell or tissue being microscope. analyzed. Exist within target - The stained DNA will appear sample. blue under a fluorescence microscope. ◼ Step V – Visualization visualization using fluorescence microscopy. Interpretation of Results MARIANO | BSMLS – 2A 6 CYTOGENETICS College of Allied Health Sciences Bachelor of Science in Medical Laboratory Science 1st Semester | A.Y. 2024-2025 Each fluorescently labeled probe that hybridizes to a cell nucleus in the tissue of interest will appear as a distinct fluorescent dot. - For Deletion Fusion Signal patterns, if it is normal, there is a ✓ Diploid nuclei will have two dots mix of red and green signals ✓ If there is duplication in the region of interest, that are fused or overlapped the gain will result in more than two dots indicating intact regions. If we ✓ If there is a loss in the region of interest, one compare it to normal break- or zero dot will result apart signal patterns, there is a ✓ If a small deletion is present in the region yellow appearance while in complementary to the probe, the probe will normal deletion fusion, there are not hybridize two distinct signals (red and ✓ If a duplication is present, more of the probe green) indicating the genes are can hybridize. not fused. There is no fusion. - In Abberant Break-apart Signal pattern, there are two separated signals (1 red, 1 green), indicating there is a chromosomal break-apart whereas, in Abberant Deletion Fusion, the green signals combine which indicates that genes have been joined together. - For 3 Color Break-apart Signal - For deletion signal patterns, if Patterns, if it is normal, it involves we compare to normal a combination of three different deletion, there are two pairs of colors (red, green, blue or signals, 1 green and 1 red, yellow) which are very close to indicating there is no deletion. each other. In Aberrant, three While in the Abberant Deletion, colors are separated which only 1 set of red signal is indicates a break or observed, there is a missing rearrangement involving color which suggests a deletion multiple chromosomal regions. in one copy of chromosome. - For Translocation Dual Fusion - For Break-apart Signal patterns, Signal Patterns, if it is normal, 2 if it is normal, there are two sets pairs of green and red signals of overlapping signals are are present indicating that no present appearing yellow (red translocation has occurred. In and green signals) due to the Aberrant, the separation of close proximity of the two colors green and red signals indicates thus indicating there is no a translocation event resulting in chromosomal break apart. the fusion of chromosomal While in Abberant Break-apart patterns. signal patterns, the green and red signals are separated thus indicating there is a chromosomal break. Stringency Conditions and Controls - For Amplification Signal Patterns, 2 pairs of red and green signals Stringency Conditions: temperature, salt are present representing normal concentration, and other factors that affect unaltered chromosome radios. the stability of the hybridization reaction But for Abberant Amplification Controls: patterns, multiple red signals are - Positive controls, such as present which indicate probes known to hybridize to amplification or increase in the specific regions number of copies of the target DNA sequence. MARIANO | BSMLS – 2A 7 CYTOGENETICS College of Allied Health Sciences Bachelor of Science in Medical Laboratory Science 1st Semester | A.Y. 2024-2025 - Negative controls, such as 3. Due to Failure to detect signal FISH is higher probes should not hybridize to sensitive for trisomy but less sensitive foe the target. detecting chromosome loss or deletion. 4. Cytogenetic data can be obtained only for the target chromosomes thus FISH is not a good screening tool for cytogenetically heterogeneous disease. 5. Requires fluorescence Microscopy and an image analysis system. Hybridizer Applications of FISH Diagnostic: ◼ The identification of specific chromosome Fluorescent Microscope abnormalities. ◼ The characterization of marker chromosomes. ◼ Interphase FISH for specific abnormalities in cases of failed cytogenetics. ◼ Monitoring disease progression ◼ Monitoring the success of bone marrow transplantation. Research: ◼ The identification of new non-random abnormalities (by M-FISH or SKY). ◼ Gene mapping COMPARATIVE GENOMIC HYBRIDIZATION (CGH) ◼ Identification of regions of amplification or deletion by CGH. is a molecular cytogenetic method for ◼ The identification of translocation analyzing copy number variations with the breakpoints help of hybridization technique. ◼ The study of 3D chromosome organization in interphase nuclei. - Used for the analysis of gains or losses in the DNA content of test Advantages of FISH samples - First described in 1993 by ✓ Rapid technique and large number of cells can be scored in a short period. Kallioniemi and colleagues. ✓ Efficiency of Hybridization and deletion is high. Sensitivity and specificity are high. ✓ Cytogenetic data can be obtained from non-dividing or terminally differentiated Principle of CGH cells. ✓ Cytogenetic data can be obtained from ◼ CGH is based on the principle poor samples that contain too few cells for of competitive fluorescence in situ routine cytogenetic analysis. hybridization (FISH). In this technique, DNA from the test and reference samples are Limitations of FISH labeled with different fluorescent dyes, typically red and green, respectively. These 1. Restricted to those abnormalities that can labeled DNA fragments are then denatured be detected with a currently available and allowed to hybridize to a metaphase probe. 2. Only one or a few abnormalities can be spread of chromosomes from a normal assessed simultaneously. individual. MARIANO | BSMLS – 2A 8 CYTOGENETICS College of Allied Health Sciences Bachelor of Science in Medical Laboratory Science 1st Semester | A.Y. 2024-2025 - Based on co-hybridization of different legal tests and reference DNA onto metaphase spreads. - The signal intensity ratios of the two fluorescent labels along the chromosome then reflect DNA copy number changes in test DNA relative to the reference DNA. - Basically, this principle explains how deletion and duplications are identified. Detecting Copy Number Variations - Deletion: using a red probe for Reference DNA; and a green ◼ CGH is particularly useful for probe for test DNA; the intensity detecting CNVs, which are variations in the of the red probe which targets number of copies of specific DNA segments. the deleted region will be These variations can range from small lowered in the test DNA bcoz deletions or duplications of a few genes to there is less target DNA in which large-scale gains or losses of entire the red probe will bind to. chromosomes. - Duplication: Since there is an extra copy of chromosome, there are many regions where in - These are alterations of the DNA red probe will bind to so the of a genome that results in the signal intensity will reflect cell having an abnormal copy an increase in red signal of cells of one or more sections compared to green signal. of DNA. The intensity of the fluorescent signals along each chromosome is then analyzed. If the test sample contains more copies of a ◼ Deletions: A deletion occurs when a particular DNA segment than the reference segment of DNA is missing from the test sample, the red signal will be stronger in that sample compared to the reference. This will region. result in a stronger green signal in the corresponding region of the chromosome. Conversely, if the test sample has fewer copies, the green signal will be stronger. Regions with equal copy numbers in both Why red signal is stronger? samples will show a yellow signal, indicating - If a segment of your DNA is a balanced hybridization. present in multiple copies in the test sample, there will be more of the target sequence of red Fluorescence Signal Interpretation probes to bind to. So, more binding sites for red probes 1. Equal signals (indicates balanced that’s why the red signal is hybridization; there are no copy variations stronger. on the regions of DNA.) 2. Increased green signal (indicates deletion) 3. Increased red signal (indicates duplication) MARIANO | BSMLS – 2A 9 CYTOGENETICS College of Allied Health Sciences Bachelor of Science in Medical Laboratory Science 1st Semester | A.Y. 2024-2025 After labeling, these two labeled DNA Why green signal is stronger? are combined with the presence of - If a segment of DNA is missing unlabeled Cot-1 DNA (placental DNA from the sample, there will be that is enriched for repetitive DNA less target sequence for the red sequence; used for blocking or probe to bind to. This means suppressing repetitive sequences which that there are more green are commonly detected in telomere probes will remain anode and centromere region). After resulting to a stronger green combining, the next process is denaturation. The reason why DNA signal in that region. needs to undergo denaturation, it’s because it prepares the DNA sample for hybridization. During denaturation, double-stranded DNA is separated into ◼ Duplications: A duplication occurs when a single strands by breaking the hydrogen segment of DNA is present in multiple copies bonds between complementary bases. in the test sample compared to the This will allow labeled test and reference reference. This will result in a stronger red DNAs to hybridize with their signal in the corresponding region of the complementary sequences on the chromosome. target DNA. Procedures of CGH The next step is hybridization, incubating the slide containing the test and reference DNA in a moist chamber for 48 - 72 hours. After hybridization, wash slide and apply counter stain which is DAPI (4’6 – diamidino – 2 – phenylindole). After counterstaining, next is the visualization. Observe slide under fluorescent microscope equipped with probe cct camera with specific optical filter sets (consist of triple-band past beams splitter, triple-band past emission filter, single-band excitations filter) WORKFLOW CGH begins with the isolation of genomic tumor DNA or test DNA and normal genomic DNA or reference DNA. After isolating the two DNAs, these two are labeled with two different fluorochrome. For genomic tumor DNA/test DNA, this will be labeled using green fluorochrome in which is the commonly used is Fluorescein Isothiocyanate (FITC). For reference DNA, we use red fluorochrome which is the tetramethyl rhodamine isothiocyanate (TRITC) to label them. MARIANO | BSMLS – 2A 10 CYTOGENETICS College of Allied Health Sciences Bachelor of Science in Medical Laboratory Science 1st Semester | A.Y. 2024-2025 Applications of CGH Array CGH: A High-Resolution Alternative/Chromosomal Microarray Analysis or ◼ This technology was first developed as a Microarray-Based Comparative Genomic research tool for the investigation of Hybridization genomic alterations in cancer. ◼ Array CGH (aCGH) is a more advanced ◼ It allows for a high-resolution evaluation of version of CGH that uses DNA DNA copy number alterations associated microarrays instead of metaphase with chromosome abnormalities. chromosomes. Microarrays contain thousands or millions of DNA probes that are ◼ It provides clinicians with a powerful tool to designed to target specific regions of the use in their increasingly sophisticated genome. This allows for a much higher diagnostic capabilities. resolution, typically in the range of 100 kilobases or less. ◼ The use of CGH is considered EXPERIMENTAL ◼ aCGH has become the gold standard for AND/OR INVESTIGATIONAL when performed detecting CNVs in clinical and research in the absence of symptoms or high-risk settings. It offers several advantages over factors for a genetic disease or when traditional CGH, including: knowledge of genetic status will not affect Higher resolution: aCGH can detect treatment decisions or screening for the smaller CNVs and those located closer disease. together. Increased sensitivity: aCGH is more ◼ Screening for prenatal gene mutations in sensitive than traditional CGH, meaning fetuses without structural abnormalities or it can detect CNVs that may be missed testing products of conception after AI. by traditional methods. (Artificial Insimination) – the focus is slightly Automation: aCGH is highly on rare genetic testing via methods like non- automated, which reduces the risk of invasive prenatal testing to detect human error and allows for faster mutations processing of samples. ◼ Diagnosis of melanoma. ◼ It helps in the detection of balanced rearrangements of chromosomes and for the comparison of normal and suspected DNA samples. Limitations of CGH ◼ While CGH is a powerful tool for detecting CNVs, it has some limitations: Resolution: Traditional CGH has a relatively low resolution, typically in the range of 5-10 megabases. This means that it may not be able to detect small - The test and reference DNA are CNVs or those located close together. labeled with fluorescent dyes - CGH cannot detect and applied to the microarray. translocations and inversions. Then, these 2 DNAs are Balanced rearrangements: CGH cannot detect balanced chromosomal rearrangements, such as hybridized to your microarray. translocations or inversions, because these do not Then, the microarray scanner alter the copy number of DNA segments. measures fluorescent signals and the computer software Technical challenges: CGH requires specialized analyzes the data and equipment and expertise, and the interpretation of generates a plot. results can be complex. - In aCGH, we don’t need to undergo denaturation. MARIANO | BSMLS – 2A 11 CYTOGENETICS College of Allied Health Sciences Bachelor of Science in Medical Laboratory Science 1st Semester | A.Y. 2024-2025 Spectral Karyotyping (SKY) ◼ Spectral karyotyping is cytogenetical technique used to simultaneously visualize all the pairs of chromosomes in an organism in different colors. ◼ The sky technique is useful for identifying chromosomal abnormalities. ◼ We can arrange the chromosomes according to their number just by visualization of different colours aquire by the chromosomes. Application of SKY ◼ This technology allows the isolation of structural chromosome abnormalities ◼ which then allows determination of the precise molecular address of chromosome breakpoints associated with deletions translocations and insertion. ◼ SKY can discern the aberrations that can’t be detected very well by conventional banding technique and Fluorescent in situ hybridization (FISH). ◼ It allows visualization of all chromosomes in different colours on same platform which is very easy to detect chromosomal abnormalities. CONCLUSION ◼ Chromosomal genetic abnormalities are the hidden cause for the huge economic losses. ◼ Molecular cytogenetic techniques like FISH, CGH and SKY are the available advanced diagnostic tools to detect such chromosomal abnormalities and to prevent the spread in the population which indirectly avoid the economic consequences. MARIANO | BSMLS – 2A 12