Cytogenetics - Lesson 8

FISH

Fluorescence In-Situ Hybridization

Full name of FISH

FISH

Cytogenetic technique that uses fluorescent probes that bind specifically to a part of chromosomes complimentary to its sequence

FISH

Useful in detecting and mapping the presence or absence of particular DNA sequences within chromosomes

Specific probes

Rapid diagnosis of trisomies and microdeletions is acquired using ____

Metaphase FISH

Gold standard and routinely done on cultured cells

Metaphase FISH

Allows direct visualization of chromosomes and exact position of signals

Metaphase FISH

Useful in detection of structural changes in genome

Interphase FISH

May also be done on uncultured specimens

Interphase FISH

Advantageous in the rapid screening of many nuclei for prenatal diagnosis and newborn studies

Interphase FISH

Beneficial in the study of samples with a low mitotic index such as most solid tumors

Interphase FISH

Inability to detect unknown structural chromosomal changes

1. Amniocytes

2. Chorionic villious cells

3. Lymphocytes

4. Cells from bone marrow aspirates or solid tumors

5. Fibroblasts

Samples for Metaphase FISH

1. Amniocytes

2. Peripheral blood smears

3. Bone marrow aspirate smear or direct harvest

Samples for Interphase FISH

Amniocytes

Specimen for ploidy analysis during prenatal studies in Interphase FISH

Peripheral blood smears

Specimen for ploidy analysis in newborns in Interphase FISH

Bone marrow aspirate smear or direct harvest

Specimen for translocation or copy number analysis in cancer studies in Interphase FISH

FISH Probes

Complementary sequences of target nucleic acids tagged or labeled with fluorophores

20 to 1000 base pairs

FISH Probes size range

Direct Labeling

Fluorophores are directly attached to the probe

Direct Labeling

Less sensitive type of FISH Probe labeling

Indirect Labeling

Chemical conjugation of the nucleic acid with a nonfluorescent molecule that can bind fluorescent material after hybridization

• FITC

• Rhodamine

• Cyanines

Examples of Direct Labeling

• Biotin

• Digoxigenin

Examples of Indirect Labeling

Locus Specific Probe

Binds to a particular region of a chromosome

Locus Specific Probe

Used when only a small portion of a gene is isolated and want to determine on which chromosome the gene is located or how many copies of a gene exist within a particular genome

Single Color

Designed to cover a gene of interest in Locus Specific Probe

Dual Color

Designed to cover any 2 genes for the detection of any aberrations in Locus Specific Probe

Dual Color

Allows simultaneous detection of numerical abnormalities of two to three regions in one FISH assay in Locus Specific Probe

Alphoid or Centromeric Repeat

Generated from repetitive sequences found in the middle of each chromosome

Alphoid or Centromeric Repeat

Used to determine whether an individual has the correct number of chromosomes or if there is aneuploidy in the patient’s genome

Short Tandem Repeat Polymorphism

Can be detected by Alphoid or Centromeric Repeat Probe

Subtelomere Probe

Specific to the subtelomere region of chromosome

Deletions and Rearrangements

What Subtelomere Probe detects

Whole Chromosome Probe

Collection of smaller probes that bind to the whole length of chromosome

Whole Chromosome Probe

Useful in the examination of chromosomal aberrations

Pre-Natal FISH Probe

Comprise of different combinations of fluorophore-labeled probes specific for chromosomes 13, 18, 21, X, and/or Y

Formamide (salt)

Probe and target DNAs are denatured using high temperature incubation in a _____ solution

Stringent washing

Nonspecific binding is eliminated via ___

1. Detection and characterization of chromosome

2. Detection and analysis of prenatal chromosomal abnormalities

3. Study of chromosomal abnormalities associated with cancer

Application of FISH

Comparative Genomic Hybridization (CGH) on Metaphase Cells

Technique that uses DNA from the cells of interest, rather than using a standard karyotype, for chromosomal analysis

Comparative Genomic Hybridization (CGH) on Metaphase Cells

Useful especially in some cancers when only DNA is available rather than any growing cells

Comparative Genomic Hybridization (CGH) on Metaphase Cells

Used successfully for clinical analysis particularly with cases that have a low (or no) mitotic index

Comparative Genomic Hybridization (CGH) on Metaphase Cells

Not useful for detecting balanced rearrangements

Multiplex FISH (M-FISH)

Technique that allows the investigator to view a karyotype so that each chromosome is “painted” with a different color

Multiplex FISH (M-FISH)

Ratio-labeled probes are used to create a distinct computer-generated false color for each chromosome

Multiplex FISH (M-FISH)

Useful for complex rearrangements like those seen in neoplastic disorders and solid tumors

Multicolor Banding (mBAND) Analysis

Uses chromosome-specific mixtures of partial chromosome paints that are labeled with various fluorochromes

Multicolor Banding (mBAND) Analysis

A computer program analyzes metaphase chromosome data and produces a pseudocolored, banded karyotype with an estimated resolution of 550 bands, regardless of chromosome length

Multicolor Banding (mBAND) Analysis

Advantageous for the determination of breakpoints and the analysis of intrachromosomal rearrangements and can be particularly useful in preparations with shorter chromosomes

Fiber FISH

Technique that is almost entirely used for research

Fiber FISH

Probes are applied and can be physically ordered on the fibers

Fiber FISH

Allows the chromosomes to be stretched out and elongated

Fiber FISH

Provides a much higher spatial resolution and allows for correct orientation and placement of probes and for precise mapping of probes

Primed In Situ Labeling (PRINS)

PCR on a slide

Primed In Situ Labeling (PRINS)

Primers of interest are hybridized on a slide and then subjected to cycles of denaturation, reannealing, and elongation that are used to incorporate labeled nucleotides

The labels are then detected fluorescently or labeled nucleotides are incorporated during the reaction

Primed In Situ Labeling (PRINS)

Can differentiate hybridization with the alpha satellite sequences for chromosomes 13 and 21, something that cannot be done with traditional FISH

Reverse FISH

Used to identify material of unknown origin

Reverse FISH

This unidentified material, such as a marker chromosome or duplication, is flow sorted or microdissected off of a slide after G-banding

The DNA from this material is extracted, PCR-amplified and labeled with a fluorochrome

This is then used as a probe and hybridized to normal or patient metaphase chromosomes to identify the origin of the unknown material

Copy Number Variations

Alterations of the DNA of a genome resulting in the cell having an abnormal number of copies of one or more sections of the DNA

Tumorigenesis

Amplifications and deletions can contribute to ___

Amplification

Most common change seen in malignancies

Comparative Genomic Hybridization (CGH) or Chromosomal Microarray Analysis (CMA)

Molecular-cytogenetic method for analysis of copy number changes in the DNA content of a given subject’s DNA and often in tumor cells

Kallioniemi in 1993

First described CGH/CMA

Dual

Number of probes CGH uses

Metaphase

CGH/CMA is hybridized to ___ chromosomes

1. Array

2. Matrix

Kinds of CGH

Charged Coupled Device (CCD) Camera

Fluorescent microscope equipped with a ____

Peripheral Blood Lymphocytes

Where the blood sample comes from for CGH

3 to 10 Mb

Resolution of CGH

Fluorescein Isothiocyanate

FITC

Green

Color of FITC

Tetramethylrhodamine Isothiocyanate

TRITC

Red

Color of TRITC

Cot 1 DNA

Blocks repetitive DNA sequences and prevents nonspecific hybridization

Human male placenta

Where cot 1 DNA is derived from