Mutation detection techniques

Karyotyping

• Direct observation of metaphase chromosome structure by arranging metaphase chromosomes according to size

• Widely used to detect chromosomal abnormalities; whether numerical or structural abnormalities

• Can detect genome mutations or aneuploidy

Karyotype

is the complete set of chromosomes in a cell

GTG Banding (G-bands trypsin Giemsa) (G banding)

• The most commonly used banding and staining method

• Produces a unique combination of a dark like-G-negative permits G-positive bands recognition and that of all individual 23 chromosome pairs for analysis

TRUE OR FALSE:

Karyotyping

A method wherein cell division is simulated by mitogen

Metaphase

What phase is cell division in karyotyping arrested?

Colcemid

This stops cell division at metaphase

Potassium chloride

A hypotonic treatment that swells the cell but avoids excess exposure as it may cause rupture of the cells

TRUE

TRUE OR FALSE: Humans have 46 chromosomes or 23 pairs which are classified Autosomes or Chromosomes 1-22

TRUE

TRUE OR FALSE: Chromosome 1 will be largest and Chromosome 22 will be the smallest

46XX

Normal karyotype of females

46XY

Normal karyotype of males

• short arm (P-Arm)

• long arm (Q-Arm);

The centromere divides the chromosome into two:

TRUE

TRUE OR FALSE: A plus (+) or minus (-) sign before a chromosome indicates an extra or missing chromosome respectively

Triploidy or Tetraploidy

with 3 or 4 copies of each chromosome

Aneuploidy

deviation from the euploid number that represents positive (gain) or negative (loss) of a specific chromosome

• Monosomy

• Trisomy

2 major forms aneuploidy

Monosomy

• loss of one chromosome

• Example: Turner Syndrome (monosomy X: 45X or 45XO chromosome)

Trisomy

an addition of one chromosome

• Translocations

• Deletion

• Inversion

• Isochromosome

• Insertion

• Ring Chromosome

Structural chromosomal abnormalities:

Fluorescence In Situ Hybridization

• Widely used to detect protein and RNA as well as DNA structures in place in the cell, or in situ

• More rapid assay with higher resolution and flexibility than karyotyping

TRUE

TRUE OR FALSE: FISH target specific sequences of chromosomes using fluorescent probes

Fluorescence In Situ Hybridization

• Requires fluorescence microscope that will excite fluorescent emission for the probes and special filters for detection of fluorescence emitting in different wavelengths

Fluorescence In Situ Hybridization

Determines copies of segment of DNA that are present or absent in a cell

Fluorescence In Situ Hybridization

Form of DNA testing for genetic diagnosis in which a special region of a chromosome is stained with a fluorescent dye

Fluorescence In Situ Hybridization

Used to localize and detect specific mRNA, (in preserved tissue sections or cell preparations), sequences by hybridizing the complementary strand of nucleotide probe to the sequence of interest

1. Probe selection

2. Probe generation

3. Probe labeling

4. Fixation of tissue

5. Hybridization and washing

6. Detection

7. Observation

Steps in FISH:

• ssDNA

• dsDNA

• sscRNA

• Synthenic oligonucleotides

Probes that can be used in FISH:

Nick Translation

What type of probe generation:

• where the DNAs creates dsDNA and DNA Polymerase 1 replaces nucleotides with labeled ones

PCR Amplification using tagged nucleotides

What type of probe generation:

• uses smaller probes that amplifies limited DNA sources, the DNA polymerase is used for genome amplification to create high quality probes

• 3H

• 32P

• 35S

• 14C

• 125I

Radioisotopes:

• Biotin

• Digoxigenin

Non-radioactive labels:

• Acetic Acid-Alcohol Mixture

• Glutaraldehyde

• Paraffin & Formalin

• 4% Paraformaldehyde

Fixatives that can be used in FISH:

4% Paraformaldehyde

most widely successful fixative solution, this provides satisfactory compromise between the variable and good sensitivity

Acetic Acid-Alcohol Mixture

best probe penetration that permit the loss of RNA from tissue

Glutaraldehyde

provide best RNA retention and tissue morphology but extensive protein cross-linking makes the probe penetration low

Paraffin & Formalin

increased cross-linking or loss of mRNA hence decreased sensitivity

Hybridization and washing

What step in FISH:

• Denaturation of the DNA is obtained by heating the DNA which separate the two strands and allow access of the single strand

• performed by placing a small amount of solutions containing the hybridization probe on a cover slip, which is then placed on the slide containing tissue sections to incubate overnight

• washes are serially applied to the slides to remove the probe that is not bound to target DNA/RNA

Denaturation of the DNA

In FISH, this is obtained by heating the DNA which separate the two strands and allow access of the single strand

Hybridization

is performed by placing a small amount of solutions containing the hybridization probe on a cover slip, which is then placed on the slide containing tissue sections to incubate overnight

• Probe construction

• Temperature

• pH

• Formamide

• Salt concentration

Parameters that play a crucial role in specificity of target labeling during hybridization and washing:

What step in FISH

• Final step of FISH

• Recognizing the probes with fluorescent antibodies corresponding to antigens incorporated in the probes

• Expensive

• Time consuming

• Limited ability to precisely define which genes and breakpoints are involved

Limitations of FISH

Comparative Genome Hybridization (CGH)

Can detect intrachromosomal amplifications or deletions

Comparative Genome Hybridization (CGH)

Has the ability to identify the locations of deletions or amplifications throughout the genome

• Cyanide 3

• Cyanide 5

Two colorimetrically distinct dyes used in CGH

Cyanine Dye 3

fluoresces at a wavelength of 550 nanometers is often represented as green

Cyanine Dye 5

• fluoresces at the far red region of the spectrum of a wavelength of 650-667 nm is represented as red.

• Derivatives of this dye which releases the red orange region is also available

TRUE

TRUE OR FALSE: Cyanide 3 and 5 are water-soluble

Comparative Genome Hybridization (CGH)

involves the isolation of DNA from two sources compared most commonly a test or reference source

gain of material

In CGH, a higher intensity of the test sample color in a specific region of the chromosome indicates?

loss of material

In CGH, a higher intensity of the reference sample indicates?

no difference between the two samples in that location

In CGH, neutral color (yellow) indicates

Comparative Genome Hybridization (CGH)

Can only detect gains or losses relative to that ploidy

Comparative Genome Hybridization (CGH)

Can also be used to study chromosomal aberrations in fetal and neonatal genomes

Comparative Genome Hybridization (CGH)

Is not able to detect structural chromosomal aberrations without copy number changes, such as mosaicisms, balanced chromosomal translocations and inversions is one of limitations of CGH

TRUE

TRUE OR FALSE: some mostly inherited disease associated sequence changes in DNA occur frequently at the same genetic location

TRUE

TRUE OR FALSE: Detection of mutations in large genes requires screening across of thousand base pairs to detect a single altered nucleotide

Whole gene sequencing

is a complex approach especially with regard to interpretation

• Enzyme immunoassays

• Immunohistochemistry

• High Performance Liquid Chromatography

• Gas Chromatography

• Mass spectrometry

Biochemical methods:

Biochemical methods

• This type of testing is useful for metabolic defects in which several genes are involved in the disease.

• Phenotype and actual protein or amino acid alterations can also be detected

immunoassays

• detect the presence of hormones, drugs, antibodies, cancer biomarkers, and other metabolites in blood, urine, or other biological fluids.

Enzyme immunoassays (EIA)

• Are flexible, potentially high-throughput methods for detecting a variety of analytes.

• These methods involve the use of specific antibodies or other ligands to detect the presence of the target molecules.

Enzyme immunoassays (EIA)

• Formats for direct antigen detection.

• Antibodies specific for the target analyte are immobilized in plate wells.

Enzyme immunoassays (EIA)

plate wells, strips, or capillaries coated with capture antibody are exposed to a test fluid, usually serum, plasma, or urine that is diluted in the appropriate assay buffer.

Alkaline phosphatase or horseradish peroxidase

In EIA, after rinsing away unbound material, detection antibodies covalently linked to — are introduced

Immunohistochemistry

Has been a standard in pathology for many years. It provides the advantage of integrating target detection, localization, and quantification in the context of tissue morphology.

Immunohistochemistry

Targeted therapeutic agents have increased the use of this biochemical method to assess tissue expression of the target molecules. These guide treatment strategies at a relatively low cost compared with other methods.

TRUE

TRUE OR FALSE: In immunohistochemistry, a tissue sample biopsy is fixed, embedded, and cut into 10 sections, then treated with antibodies that bind to specific markers.

visual staining enzyme or dye

In IHC, what is applied to identify the binding under a microscope?

fluorescent or colorimetry

In IHC, signal can be?

TRUE

TRUE OR FALSE: In IHC, to generate the signals, antibodies are attached to covalently fluorescent molecules or enzymes, such as horseradish peroxidase alkaline phosphatase.

Fluorescent signals

the fluorescent molecules attached to the bound antibodies will emit signals when the floor is excited

colorimetry signals

a substrate solution is added to the bound antibodies. The substrate isoxidized by horseradish peroxidase or alkaline phosphatase, leading to a color reaction.

direct antibody staining

• the floors of enzymes are attached to the antibody that directly binds to the target molecules in the tissue, the primary antibody.

• This method is faster than the alternative indirect staining, but it has limited signal intensity.

indirect staining

• The primary antibody is not attached to the signaling molecule.

• A second or secondary antibody binds to the primary antibody, usually from a different species, such as rabbit, mouse, or goat, that carries the signal.

• may require additional blocking with unlabeled antibodies or IgG protein to avoid non-specific binding to the secondary antibodies.

High Performance Liquid Chromatography (HPLC)

is an advanced analytical technique used to separate, identify, and quantify components in a liquid mixture based on their affinity for stationary and the mobile phase.

High Performance Liquid Chromatography (HPLC)

It uses high pressure to pump a liquid solvent at the mobile phase through a column packed with absorbent material in the stationary phase.

High Performance Liquid Chromatography (HPLC)

is the basis for separation or analysis instruments such as amino acid analyzers.

• Mobile phase (Solvent)

• Stationary phase (Solvent support)

HPLC consists of two phases:

Gas chromatography

• is the separation of a vaporized sample through a column of inner carrier gas and liquid stationary phase that molecules. differentially

• The sample is injected through the injection port and then separated molecules are recorded by the detector.

Mass spectrometry

In this automated method, an ion source sends high-energy electrons that hit the target sample molecules, separating them into ions, usually cut ions with the loss of one or two electrons.

mass spectrometer

converts molecules to ions that can be moved in a magnetic field based on their charge and mass.

• single-strand conformation polymorphism

• heliostatic hybridization

• heteroduplex technology

• melt curve analysis

• oligomer analysis, and array

Hybridization-based methods:

Hybridization-based methods

will detect specific DNA or RNA sequences by enabling sequelstranded labeled probes to anneal with the complementary target sequences, forming a double-stranded DNA hybrids. stable

Sequence polymerization-based methods

• Utilize DNA enzymes to synthesize new DNA strands, allowing for the identification, amplification, and sequencing of genetic material.

• These techniques rely on the base-pairing rules, A to T, G to C, to either determine the order of nucleotides or amplify a specific DNA sequence for detections.

• Sequence-specific PCR

• Allelic Discrimination with Fluorogenic probes

Sequence-based methods

Enzymatic or chemical cleavage methods

are fundamental to nucleic acid analysis for structural mapping, sequencing, and genetic mapping.

• Restriction fragment Length Polymorphisms

• Non-isotopic RNase cleavage assay

• Cleavase assay

Enzymatic or chemical cleavage methods: