Cytogenetics - Lesson 7

Karyotyping

Karyotype

Number and appearance of chromosomes

1. Size of chromosome

2. Position of chromosome

3. Presence of secondary constrictions

4. Size of satellites

Information from a Karyotype

Karyology

Study of whole sets of chromosomes

Idiogram / Karyogram

Standard format of representing chromosomes as diagram when haploid set of chromosomes of an organism are ordered in a series of decreasing size

Asymmetric Karyotype

Show larger differences between smaller and larger chromosomes in a set

Asymmetric Karyotype

Have more acrocentric chromosomes and relatively advanced features

Symmetric Karyotype

Show lesser differences between smaller and larger chromosomes in a set

Symmetric Karyotype

Have more metacentric chromosomes and no advanced feature

GA Levitzky

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

Crepis

Genus that was carefully studied on karyotype asymmetry

Compositae

Family that was carefully studied on karyotype asymmetry

Degree of Asymmetry

Proportion of metacentric and acrocentric chromosomes and ratio between largest and smallest chromosomes in a set

Heterochromatin

Methylation of DNA and histones causes nucleosomes to pack tightly together

Heterochromatin

Transcriptions factors can’t bind the DNA

Euchromatin

Histone acetylation results in loose packing of nucleosomes

Euchromatin

Transcription factors can bind the DNA

Short Term Lymphocyte Culture

Step 1 in Karyotyping

Harvesting of Lymphocytes

Step 2 in Karyotyping

Fixing the Cells

Step 3 in Karyotyping

Making Chromosome Slides

Step 4 in Karyotyping

Chromosome Analysis

Step 5 in Karyotyping

Streptomycin and Penicillin

Antibiotics that are added in the lymphocyte culture in Karyotyping

Phytohemagglutinin (PHA)

Reagent that induces mitotic activity

37 degrees Celsius for 3 days

Temperature and length for growth of culture blood cells

Logarithmic Phase

Cells must be in _______ because splitting of a cell line 2 days and changing the medium 1 day before harvest stimulates cell proliferation significantly

Colcemid or Colchicine

Reagent that arrests cell cycle at metaphase stage

Hypotonic Solution (KCl or Sodium citrate)

Where the cell pellet is resuspended before it is mixed

Actinomycin D or Ethidium bromide (before harvesting) or bromodeoxyuridine (BrdU) before colcemid

Additional modifications that allow for enrichment of long chromosomes use ____

Absolute methanol:glacial acetic acid, 3:1 or Carnoy’s fixative

Fixative solution added to cells to be treated

GTG-banding (G-bands by Trypsing using Giemsa)

Stain of the Karyogram

Trypsin

Digest chromosomes at regions rich in basic amino acids

Arginine and Lysine

Rich amino acids digested by Trypsin

Cytovision by Applied Imaging Inc.

Computer software program that analyzes the Karyogram

International System of Human Cytogenetic Nomenclature (ISCN)

Organization that sets the rules of arrangement of chromosomes according to size and banding patterns

Paris Report (1971)

First attempt to provide nomenclature for chromosome banding

Caspersson (1958)

First author to publish a paper describing the use of quinacrine mustard to stain chromosomes

Band

Part of chromosome clearly distinguishable from its adjacent segments by appearing darker or lighter

Quinacrine (QTQ)

Stain of Q-banding

Giemsa (GTG) and Wrights

Stain of G-banding

Giemsa (RHG) and CH3/DA

Stain of R-banding

Giemsa (CBG)

Stain of C-banding

AgNO₃

Stain of NOR banding

Q (Quinacrine)

R (CH3/DA)

Replication banding (Hoechst)

DA-DAPI staining

Banding types that use a Fluorescent Microscope

UV light

Quinacrine is subjected to ___

AT

Quenches dye and fluorescence in Quinacrine

GC

Quenches dye but don’t fuorescence in Quinacrine

AT

In Quinacrine, it appears dark and situated in heterochromatin

GC

In Quinacrine, it appears light and situated in euchromatin

Quinacrine

Simple and versatile staining

Quinacrine

Used where G band is not accepted

Quinacrine

Used in study of chromosome heteromorphism

Quinacrine

Tendency to fade during examination

Quinacrine

Photo-degradation

Quinacrine

Chromophore absorb light of a particular wavelength due to a chemical bond formed between dye and light

Quinacrine

UV light breaks chemical bond

Giemsa

Uses trypsin, urea, protease

Thiazine+ and eosin-

Interaction of DNA with ___ and ___ components of Giemsa stain brightens sulfur-rich regions

• Methylene Azure+

• Methylene Violet+

• Methylene Blue+

Dyes of Giemsa

Giemsa

Used in identification of bands rich in sulfur

Giemsa

Used in identification of chromosomal abnormalities

Giemsa and Centromeric

Used in gene mapping

Giemsa

Not used in plants

NOR

Staining where chromosome is air dried

5% trichloroacetic acid

In NOR, there is treatment with ____ at 95 degrees Celsius for 30 minutes

0.1 N HCl

In NOR, there is treatment with ____ at 60 degrees Celsius for 30 minutes

NOR

Banding pattern is structural non-proteins linked to ____ (histone region)

NOR

Used in identification of nucleolar organizer region

NOR

Superior banding pattern of plants

Summer in 1971

First used Giemsa

Matsui and Sasaki in 1973

First used NOR

Centromeric

Treated with alkali solution denaturing DNA

Centromeric

Washed with sodium citrate at 60 degrees Celsius for 30 minutes for repetitive DNA renature but unique DNA don’t renature

Centromeric

Stained with Giemsa solution

Centromeric

Banding pattern at heterochromatin region (AT)

Centromeric

Identification of chromosomes in insects and plants

Centromeric

Identification of bivalents at diakineses using both centromere position

Centromeric

Used in paternity testing

R-banding

Opposite of G-banding

ptel and qtel

In chromosomes, ends are labeled ___ and ___

Linde and Laursen in 1978

First used Centromeric banding

1

3

16

19

20

Metacentric chromosomes

2

4

5

6

7

8

9

10

11

12

17

18

X

Submetacentric chromosomes

13

14

15

21

22

Y

Acrocentric chromosomes

Heterochromatic

In C-banding, it stains ____ regions close to centromeres

Centromeric

Usually stains the entire long arm of Y chromosome