[CYTOGENETICS] Karyotyping

Karyotyping

is the number and appearance of chromosome in the nucleus of a eukaryotic cell

Karyotyping

The process of Pairing and Ordering of chromosomes

Metaphase

Phase of cell division where chromosomes are studied

bright field microscope or fluorescent microscope

two microscopes used in karyotyping

Size of chromosome

> Compare sizes of Chromosome 1 and Y chromosome

Position of centromere

> Acrocentric, Metacentric etc

Presence of secondary constrictions

Size of satellites

What are the 4 things we can get from Karyotyping

Karyotype

comes from the Greek word "Karyon," which means nucleus.

Karyology

The study of whole sets of chromosomes.

Idiogram or Karyogram

The standard format of representing chromosomes as a diagram is when the haploid set of chromosomes of an organism is ordered in a series of decreasing size.

Idiogram or Karyogram

Chromosomes are arranged from largest to smallest

Manually take pictures using a camera first

Asymmetric Karyotype

show larger differences between smaller and larger chromosome in a set.

Have more acrocentric chromosomes and relatively advanced feature.

Symmetric Karyotype

show lesser difference between smaller and larger chromosome in a set. Have more metaphase chromosomes and no advanced feature.

Asymmetrical

more complex; more asymmetrical

Describe the karyotype of humans and animals

GA Levitzky

a Russian scientist suggested that in flowering plants there is a predominant trend towards karyotype asymmetry.

This trend has been carefully studied in the genus Crepis of the family compositae

Crepis

Compositae

Species of flowering plants used by GA levitzky

Green-top vacutube (heparin)

note: EDTA citrate can interfere with chromosomes

What type of tube is used for the patients blood after harvesting

1. SHORT TERM LYMPHOCYTE CULTURE

2. HARVESTING OF LYMPHOCYTES

3. FIXING THE CELLS

4. MAKING THE CHROMOSOME SLIDES

5. SLIDE ANALYSIS

5 major steps of Karyptyping

SHORT TERM LYMPHOCYTE CULTURE

The collected blood will be grown in vitro by adding cell culture growth medium, fetal bovine serum, antibiotics, and phytohemagglutinin (PHA) - the reagent that induces mitotic activity

The cultured blood cells will be grown at 37 °C incubator for 3 days

Note that the cells must be in logarithmic phase because splitting of a cell line 2 days before harvesting, and changing the medium 1 day before harvesting, stimulates cell proliferation significantly.

phytohemagglutinin (PHA)

fetal bovine serum

antibiotics or antifungals

1) SHORT TERM LYMPHOCYTE CULTURE

the reagents that induces mitotic activity

3 components of the growth medium

HARVESTING OF LYMPHOCYTES

Addition of pre-warmed colcemid (also known as colchicine), the reagent that arrests the cell cycle at metaphase stage, into the culture and incubate for 15 mins.

Optimal exposure time to colcemid requires a balance between proliferative activity index of cells and concentration of colcemid

Centrifuge the tube at 1000 RPM for 10 mins and the cell pellet was resuspended in warm hypotonic solution (can be KCl or sodium citrate) and the solution was mixed.

Incubate at room temperature for 15 mins.

Colcemid or Colchicine

(2) HARVESTING OF LYMPHOCYTES

Used to arrest the lymphocyte cells in metaphase

1000RPm for 10mins

(2) HARVESTING OF LYMPHOCYTES

Centrifuge the tube at ______________ and the cell pellet was resuspended in warm hypotonic solution (can be KCl or sodium citrate) and the solution was mixed. Incubate at room temperature for 15 mins.

HARVESTING OF LYMPHOCYTES

additional modifications:

allow for enrichment of long (prometaphase) chromosomes by using Actinomycin D or ethidium bromide (added before harvesting), or bromodeoxyuridine (BrdU), added before colcemid treatment.

Cell synchronization can significantly increase the total yield of metaphase chromosomes. Cells are arrested at S phase by adding an excess amount of BrdU overnight (16 h). After this, the block is released by washing the cells and adding thymidine for 5.5 h before colcemid treatment (Hirai et al., 1994).

Cell synchronization

can significantly increase the total yield of metaphase chromosomes. Cells are arrested at S phase by adding an excess amount of BrdU overnight (16 h).

FIXING THE CELLS

The cell suspension in hypotonic state will be centrifuged for 1200 RPM for 5 mins.

The cell pellet will be treated with fixative solution (absolute methanol:glacial acetic acid; 3:1) or Carnoy’s fixative and will be centrifuged at 1200 RPM for 5 mins

The process will be repeated 3x and the final addition of fixative solution will require incubation at 4 °C for 10 mins.

methanol:glacial acetic acid; 3:1

or Carnoy’s fixative

Fixative used

Carboy's Fixative

Solution made up of methanol and glacial acetic acid

Methanol (1)

Preferred fixative for proteins (histones)

Glacial Acetic Acid (3)

Fixative of nucleic acids (DNA)

MAKING THE CHROMOSOMES SLIDE

5 or 6 cold slides will be layered next to each other in a paper towel.

2 or 3 drops of the samples will be dropped onto each slide and dry them spontaneously

The slide will be stained by GTG-banding (G-bands by Trypsin using Giemsa), the most common method of staining chromosomes for differentiation which uses trypsin that digests the chromosomes at regions rich in basic amino acids (Arg and Lys).

Trypsin

digests the chromosomes at regions rich in basic amino acids (Arg and Lys).

Giemsa Stain with Trypsin

Stain used for GTG banding

SLIDE ANALYSIS

Slides that will be chosen for analysis and visualization must be:

> Properly trypsinized chromosomes

> Clearly defined metaphase spreading

Requires a microscope with automated computer software program primarily, CytovisionTM by Applied Imaging Inc. which follows the International System of Human Cytogenetic Nomenclature (ISCN) that arrange chromosomes according to size and banding patterns

10-20mL

How much peripheral blood is drawn

Because they have the greatest nucleus to cytoplasm ratio (malaki nucleus)

Why are lymphocytes used and not RBC?

Fish and amphibians

We cannot culture RBCs because they are anucleated. However, _______ and ________ have a nucleus in the RBC

Antibiotics

Antifungal

Added to the nutritious culture medium so we can ensure ONLY the growth of Lymphocytes

Phytohemagglutinin

Reagents that stimulate the growth of lymphocytes and Removal of RBCs

Hypotonic Solution

Solution where we need to put the cultured media so cell membrane are broken and the chromosomes are exposed

Band

A ______ is a part of a chromosome that is clearly distinguishable from its adjacent segments by appearing darker or lighter with various banding methods (Paris conference, 1971)

Light Staining Area

Contains the euchromatin

Where gene expression happens

Dark Staining Areas

Contains the heterochromatins

Epigenetics

A branch of genetics that studies gene expression

"Does an organism express or use this gene?"

1958. Caspersson et al

published there first paper describing the use of quinacrine mustard to stain chromosome there by ushered in a new era of chromosome banding

The Paris Report (1971)

was the first attempt to provide nomenclature for chromosome banding in any species and thus its recommendations have been adopted to nonhuman species as well.

Banding Patterns

allows you to see smaller pieces of the chromosome, so that you could identify smaller structural chromosome abnormalities not visible on a routine analysis.

GC and At rich regions

Contitutive Heterochromatin Region

What are the basis of classification of banding techniques?

Metaphase Chromosomes

chromosomes whose size has condensed and whose diameter is increased are used for chromosome banding studies after fixing the stage

Q-BANDING

Banding technique by: CASPERSON 1958

G-BANDING

Banding technique by: SUMMER 1971

Nucleolar organization regions (N-BANDING)

Banding technique by: MATSUI AND SASAKI 1973

Centromeric (C-banding)

Banding technique by: LINDE AND LAURSEN 1978

Q-BANDING

We use UV light to visualize it

> Shorter wavelength

> Higher frequency and energy

Light staining areas are green

> G and C-rich regions

Dark staining areas are the apple green ones or the yellow-green

> Contains adenine and thymine-rich regions

Q-BANDING

Note: GC rich regions do not fluorescence

Q-BANDING

Banding technique used for revealing polymorphisms in Chromosomes 3,4,13,14,15,21,22

Quinarcine or mustard

The first ever discovered banding technique in Karyotyping (ito lang daw itatanong)

Color: Apple green

Light base or alkaline

Ideally, there is NO denaturation in Q banding, but we use this for denaturation of Q banding when needed

Apple green

Color of Quinarcine or mustard (Q-BANDING)

AT rich regions

What regions do Quinacrine fluorescent dye stains (light staining regions - green)

Fluorophores (fluorescent stains)

undergo photobleaching or quenching when exposed to light

High frequency

Higher Energy

Shorter wavelength

Describe the UV light (frequency wavelength and energy)

Visible light

Light to which the fluorescent light will be converted to

has:

longer wavelength

Lower energy and frequency

Q-BANDING

Banding used in study of chromosome heteromorphism

Nucleolar organization regions (N-BANDING)

The ability of specific chromosomes to generate ribosomal RNA

What part of the human chromosomes produces ribosomal RNA?

Nucleolar organization regions (N-BANDING)

Banding pattern is:

Dark bands

> Nucleolar Organizer Regions (NORs) – rDNA sites

Rest of the chromosome (usually not stained)

Nucleolar organization regions (N-BANDING)

Banding pattern used for PLANTS

Nucleolar organization regions (N-BANDING)

Uses: Trichloroacetic Acid and HCl

Stains: Structural non-histone proteins

AgNO3

Stain technique in Nucleolar organization regions (N-BANDING)

Nucleolar organization regions (N-BANDING)

Reveals polymorphisms and rearrangements of acrocentric chromosomes

Centromeric (C-banding)

Dark-staining areas are only the centromeres.

There is denaturation of the DNA.

> Destroys the hydrogen bonds

> The denatured parts are lightly staining.

Tendency of repetitive DNA (like centromere, if we denature it, it can naturally renature

We use a brightfield microscope

Centromeric (C-banding)

Detection of the centromere

Is there a centromere or no?

Centromeric (C-banding)

Stains the heterochromatin regions close to the centromeres

Note: The euchromatin are usually unstained

Centromeric (C-banding)

Usually stains the entire long arm of the Y chromosome

BaOH

Denaturating agent for the C-banding technique

Centromeric (C-banding)

Treated with alkali solution

Washed with Sodium Citrate

Stained with Giemsa

Sodium Citrate

Used in C banding to ensure that repetitive DNA centromeres so not renature

Centromeric (C-banding)

Identification of chromosomes, particularly in insects and plants.

Identification of bivalents at diakinesis using both centromere positions.

Paternity testing.

Gene mapping

Centromeric (C-banding)

Reveals polypormisms including heterochromatin inversions; evaluation of ring and dicentric chromosomes

GIEMSA (G-BANDING)

Required partial denaturation process using Trypsin

Loosen the bonding between the DNA and the histone

GIEMSA (G-BANDING)

Dark staining areas are AT-rich.

Light staining areas are CG-rich regions.

Targeted by acetyltransferases (CG) :: There is acetylation → increased gene expression

Wrights (uses bright field as well)

GIEMSA (G-BANDING) alternative staining technique

GIEMSA (G-BANDING)

Banding technique for simple chromosome photography

Q-BANDING

> Tendency to fade during examination; UV light breaks the chemical bond.

> Photo-degradation

> Chromopore- absorb light of a particular wavelength due to a chemical bond formed between dye and light.

Trypsin

The denaturing agent used in Giemsa or G STAINING

> Proteolytic enzyme by cracking open histones

We use a brightfield microscope

Boiling

Alternative to Trypsin if we want to denature the DNA

G Banding Techniques

Advantages

▪ Used in identification of bands rich in Sulfur content.

▪ Used in the identification of chromosomal abnormalities

▪ Gene Mapping

G Banding Techniques

Disadvantages

▪ Not used in plants.

R-BANDING

Makes use of heat to denature the proteins

The banding area is reversed

Dark: CG

Light: AT

R-BANDING

Used for visualization of ends of chromosomes and small positive R-bands

7q31.2

This indicates that the gene is on chromosome 7, q arm, band 3, sub-band 1, and sub-sub-band 2.

The ends of the chromosomes are labeled ptel and qtel

The notation 7qtel refers to the end of the long arm of chromosome 7

International System for Human Cytogenetic Nomenclature

ISCN

Closest or Proximal to the centromere

As per ISCN, Each area of chromosome given the lowest number

____________ of closest (proximal) to centromere

Highest number

As per ISCN, Each area of chromosome at tips (distal) to centromere