Molecular Biology - Lecture - 10 - Mutation Detection Techniques - Complete

Flow cytometry

Example of indirect method of detection for genome mutations or aneuploidy

Karyotyping

Example of direct method of detection for genome mutations or aneuploidy

Karyotype

Is the complete set of chromosomes in a cell.

Karyotyping

It is the direct observation of chromosome structure

Metaphase

Karyotyping is a direct observation of chromosome structure in what phase? By arranging chromosomes according to size

Karyotyping

Widely used to detect chromosomal abnormalities (numerical or structural)

Metaphase, Prometaphase

In karyotyping, cells are cultured and artificially arrested in mitosis during either of these phases

Culture

Chromosome analysis - Karyotyping

(1) This is done to cells for varying periods, depending on cell type, with or without stimulation

Hypotonic solution

Chromosome analysis - Karyotyping

(2) Incubated cells are then artificially arrested and later subjected into this solution to allow disruption of the nuclear cell membrane and proper dispersion of the chromosomes for analysis.

(3) Fixed, banded, and finally stained

G-bands trypsin Giemsa banding

The most commonly used banding and staining method

G-bands trypsin Giemsa banding

Chromosome staining method that produces a unique combination of a dark G-positive and like-G-negative bands that permits recognition of all individual 23 chromosome pairs for analysis

48-72 hours

Culture duration for collected living cells prior to chromosome analysis by karyotyping

Mitogen

Substance that simulates cell division

Phytohemagglutinin, Granulocyte colony-stimulating factor

Mitogen that can be added to cultured cells (to be analyzed by karyotyping)

Colcemid

Substance that arrests dividing cells in metaphase

Microtubule formation

Process inhibited by Colcemid

Hypotonic

Chromosomes in living cells that arrest in metaphase will yield a chromosome strand when the cell nuclei are disrupted by a ___ buffer

23

Amount of pairs of chromosomes assembled into a organized display or karyotype according to their size and centromere placement. Where Aneuploidy can be observed affecting several chromosomes or a single chromosome.

Blood, Bone marrow, Amniotic fluid, Placental tissue

Acceptable samples for Karyotyping

Mitotic spindle

Colcemid stops cell division at the metaphase when they are maximally condensed because it attaches to this structure that prevents the cell entering the anaphase

Low concentrated Potassium chloride

Used in hypotonic treatment of the cells, swelling the cell but avoids excess exposure as it may rupture the cells

Fixative solution

Karyotyping

(5) Solution that fixes the cell

Half meter

Karyotyping

(5.1) Height from which we drop the solution to make the chromosome slide, 2-3 drops of fluid on each side

Trypsin

Karyotyping

(6) Allow the slide to dry and then add ___

Giemsa stain, Microphotograph

Karyotyping

(7) Stain the slide by immersion with ___ and finally examine the slide to a ___ good spreads and construction of the G-bonded karyotype.

Microscopically, Video camera

Metaphase chromosome spreads are first evaluated ___ and their images or photographs are captured by a ___ and stored on a computer for later analysis

46

Total number of human chromosomes

23

Total number of pairs of human chromosomes

Autosomes

What kind of chromosomes are chromosomes 1-22?

Sex chromosomes

What kind of chromosome is chromosome 23?

XX

Female sex chromosome

XY

Male sex chromosome

Homologous chromosomes

Chromosomes from a metaphase spread that can be paired and arranged systematically to assemble a karyotype according to a well defined standard convention

International Human Cytogenetic Nomenclature

Well-defined standard convention that serves as a guide to systematically arrange a karyotype

Chromosome 1

Largest human chromosome

Chromosome 22

Smallest human chromosome

Total number of chromosomes, Sex chromosome constitution.

Description of a human karyotype according to the International Human

Cytogenetic Nomenclature

46XX

Normal female karyotype

46XY

Normal male karyotype

The internationally accepted system for human chromosome classification relies largely on the:

Length and banding pattern of each chromosome

Position of the centromere relative to the ends of the chromosome

Another useful distinguishing feature for human chromosome classification

Centromere

Divides the chromosome into 2.

p arm

Chromosome short arm

q arm

Chromosome long arm

+

Symbol before the number of a chromosome indicates there is an extra chromosome respectively

-

Symbol before the number of a chromosome indicates there is a missing chromosome respectively

Triploidy

3 copies of each chromosome

Tetraploidy

4 copies of each chromosome

Aneuploidy

Deviation from the euploid number that represents the positive/gain or loss/negative of a specific chromosome

Monosomy, Trisomy

2 major forms of Aneuploidy

Monosomy

Major form of Aneuploidy

The loss of chromosome

Turner syndrome

Most common example of Monosomy

45X

Turner syndrome karyotype

Trisomy

Major form of Aneuploidy

The gain of a chromosome

Down syndrome, Edwards syndrome, Patau syndrome, Klinefelter syndrome

Most common example of Trisomy

47,XY/XX,+21

Down syndrome karyotype

47,XY/XX,+18

Edwards syndrome karyotype

47, XX/XY,+13

Patau syndrome karyotype

47, XXY

Klinefelter syndrome karyotype

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

Fluorescence in situ hybridization

More rapid assay with higher resolution and flexibility than karyotyping

Fluorescence in situ hybridization

Method limited to the regions complementary to the FISH probes

Probes

In Fluorescence in situ hybridization, these are designated to hybridize to critical areas that are amplified, deleted, or transmutated, or rearranged in disease states

Epifluorescence microscope

Fluorescence in situ hybridization microscope used that will excite fluorescent emission for probes and special filters at different wavelengths

Fluorescence in situ hybridization

This method determine copies of segment of DNA that are present or absent in a cell

Fluorescence in situ hybridization

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

Fluorescence in situ hybridization

Used to detect nucleic acids by using probes that are coupled to a fluorescent dye

Fluorescence in situ hybridization

Used to localize and detect specific mRNA sequences in tissue sections by hybridizing the complementary strand of nucleotide probe to the sequence of interest

Fluorescence in situ hybridization

In this method, the probe binds only those parts of chromosomes with high degree of sequence complementarily

Probe selection

The first step of in situ hybridization

dSDNA, ssDNA, sscRNA, Synthetic oligonucleotides

Probes used in Fluorescence in situ hybridization

Probe generation

Step in Fluorescence in situ hybridization that involves Nick translation and PCR using tagged nucleotides

Nick translation

Fluorescence in situ hybridization - Probe generation

DNAse creates mixed and dsDNA and DNA polymerase 1 replaces nucleotides with labeled 1s

PCR using tagged nucleotides

Fluorescence in situ hybridization - Probe generation

Used for smaller probes or amplifying limited DNA sources, for genome amplification to create high quality probes

Probe labeling

Step in Fluorescence in situ hybridization that involves Radioisotopes and Non-radioactive labelling with Biotin and Digoxigenin

Radioisotopes, Non-radioactive labels

Fluorescence in situ hybridization - Probe labeling

Types of labels used

3H, 32P, 35S, 14C, 125I

Fluorescence in situ hybridization - Probe labeling

Radioisotopes used

Biotin, Digoxigenin

Fluorescence in situ hybridization - Probe labeling

Non-radioactive labels used

Fixation of tissue

Step in Fluorescence in situ hybridization that involves fixatives

Acetic acid-Alcohol mixture

Fluorescence in situ hybridization - Fixation of tissue

Fixative with best probe penetration but may permit the loss of RNA from tissue

Glutaraldehyde

Fluorescence in situ hybridization - Fixation of tissue

Fixative that provide best RNA retention and tissue morphology but with extensive protein crosslinking causes slow probe penetration

Paraffin, Formalin

Fluorescence in situ hybridization - Fixation of tissue

Fixative with decreased sensitivity resulting in increased cross-linking or loss of mRNA

4% Paraformaldehyde

Fluorescence in situ hybridization - Fixation of tissue

Most widely successful fixative; good sensitivity

Hybridization and Washing

Step in Fluorescence in situ hybridization that involves hybridization and washing

Denaturation

Fluorescence in situ hybridization - Hybridization and Washing

___ of the DNA is obtained by heating the DNA which separate the two strands and allows access of the single strand

Hybridization

Fluorescence in situ hybridization - Hybridization and Washing

This 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

Washing

Fluorescence in situ hybridization - Hybridization and Washing

The next day, ___ is serially applied to the slides to remove the probe that is not bound to target DNA/RNA

(Probe construction, Temperature, pH, Formamide, Salt concentration)

Detection

Final step of Fluorescence in situ hybridization

Expensive, Time-consuming, Limited ability to precisely define which genes and breakpoints are involved in imbalance

Fluorescence in situ hybridization disadvantages

Time-consuming

Fluorescence in situ hybridization disadvantage because the test requires knowledge in subcloning, in-vitro transcription, and bacterial expression

Comparative genome hybridization

Test that can detect intrachromosomal amplifications or deletions

Comparative genome hybridization

In this test, DNA is labeled and used as a probe on a normal metaphase chromosome spread

Comparative genome hybridization

Test with the ability to identify the location of deletions or amplifications throughout the genome

Comparative genome hybridization

In this test, the test DNA is isolated and labeled along with the control DNA

Cyanide 3, Cyanide 5

Two polymetrically distinct cyanine dyes used as fluorescent labels in Comparative genome hybridization

550 nm

Cyanide 3 wavelength

Green

Cyanide 3 color

650-667 nm

Cyanide 5 wavelength

Red

Cyanide 5 color