We have seen that the chromosome is condensed form of DNA. This condensation requires different histone and non-histone proteins. Each chromosome have reproducible ultrastructure.The study of chromosomes is called cytogenetics.

Fig 1: Structure of a Chromosome

The pictures of stained chromosomes of the cell can be photographed and arranged according to shape, structure and sizes to form a karyogram. When subjected to different treatments before staining, the chromosomes develop different dark and light regions in form of bands. The banding pattern can be used to identify homologous chromosome and detect different types of chromosomal rearrangements abnormalities.

Fig 2: Procedure for chromosome staining (with Giemsa)

There are various techniques to stain chromosomes and achieve different types of banding. Here we mention some of them:

1. Giemsa staining:

Giemsa is a visible light dye, which binds DNA through intercalation. Visible light dyes are more stable and capable of producing clearer bands than fluorochromes. Giemsa stain is a mixture of cationic thiazine dyes and anionic eosin dyes such as eosin Y.

Positive thiazine dye molecules are smaller and two molecules of the same quickly intercalate into the negative DNA molecule, and stains it blue. The anionic eosin molecule then binds the two thiazine molecule and stains the DNA purple. Giemsa stains the hydrophobic regions better.

There are four different types of banding techniques, which can be done using Giemsa: G-bands, R-bands, C-bands & T–banding. In each of the above mentioned staining techniques, Giemsa stain stains different regions due to difference in the pretreatment. As we all know, the histone proteins are uniformly spread through the length of the chromosomes. The non-histone proteins are spread at variable sites and responsible for loose or condensed state of different regions of the chromosomes. The loosely packed regions or euchromatin regions and tightly packed region are the heterochromatin regions. The pre-treatment processes differentially extract these proteins resulting into differently stained regions.

1. G-banding:

This is the most commonly used banding method for cytogenetic analysis using Giemsa stain. The technique was first developed by Dr. Marina Seabright in 1971.

G banding
Fig 3: Dr. Marina Seabright

(Image Source and more information on Dr. Marina Seabright here)

The cells are arrested in metaphase, swollen (made turgid), fixed, dropped and bursted. Then the chromosome spreads are air dried and chromosomes are pretreated before Giemsa staining (Fig 2; in details in previous post).

Pretreatment:

During the standard G-banding the chromosomes are mildly treated with proteolytic enzymes (trypsin) before staining with Giemsa. Standard protocol G-band staining when followed gives around 400 and 600 bands to be seen on metaphase chromosomes.

The two types of bands which are observed are

• Positive G-bands:

Positive G-bands are the darkly stained bands. These regions are hydrophobic, and favour the formation of the thiazine-eosin precipitate. The hydrophobicity is due to the hydrophobic proteins. The proteins are difficult to extract as they have more of disulfide cross-links (Fig 4). These proteins keep the regions more condensed . They form the late replicating heterochromatin and are generally AT-rich region.

Fig 4: Disulphide bond and sulfhydril group

• Negative G-bands:

The lightly stained bands are called negative G-bands. These regions are rich in GC base pairs. These are early replicating euchromatin and are less condensed. The proteins that bind these regions have more of sulfhydrils (fig 4) and are easily removed during pretreatment. These regions are less hydrophobic and less favorable for the formation of the thiazine-eosin precipitate.

Fig 5: Normal Human (Female) Karyotype. (Source: Thirumulu Ponnuraj, Kannan. (2011). Cytogenetic Techniques in Diagnosing Genetic Disorders)

2. R-banding:

This banding technique reveals the GC-rich euchromatin and produces positive bands that correspond to the negative G-bands and vice versa. This gives results reverse of the standard G-banding.

Pre-treatment:

In this technique, banding is produced by metaphase chromosomes in hot phosphate buffer (~87°C) before staining with giemsa stain. The incubation causes the denaturation of the AT regions of the chromosomes because of the low melting point of these regions (~65°C) as compared to that of the GC regions (~105°C).

R-banding is helps analyse the structure of chromosome ends, which stain light with G-banding but darker with R-Banding.

Fig 6: Chromosome 1 : G-banding, diagram and R-banding – (Image Source: Claude Léonard, Jean-Loup Huret; Atlas of Genetics and Cytogenetics in Oncology and Haematology)

3. C-banding:

C-banding stains constitutive heterochromatin which is present around the centromeres of all human chromosomes, and is most abundant around the centromeres of human chromosomes 1, 9, 16 and the distal long arm of the Y-chromosome.

Pre-treatment:

The pretreatment involves three successive steps; treatment with acid (HCl), followed by a alkaline treatment (barium hydroxide) and finally treatment with hot salts [saline- sodium citrate (SSC)]. Treatment with acid (HCl) brings about the removal of the purines. The alkaline treatment (barium hydroxide or Sodium borohydride) reduces the apurinated sugars. Chain breakage of the depurinated sites occur during treatment with hot salts solution [60°C, saline- sodium citrate (SSC)]. In this final treatment, the sites with highly repetitive sequence resist the breakage and get renatured. Also the sites with proteins having strong interaction are protected. Therefore, the sites protected by protein or having highly repetitive sequences like centromeres get stained.

Fig 7: (a) the G-banded karyotype of a male Bradypus torquatus (2n = 50); (b) C-banded karyotype of a male Bradypus torquatus. (Image Source: Azevedo N et. al. (2012). BMC evolutionary biology. 12. 36.)

T-Banding:

T-banding involves the staining of telomeric regions of chromosomes. The chromosomes (slides) are incubated in a phosphate or PBS buffer at 87°C followed by staining with Giemsa solution or acridine orange(OA) . T-bands are heat-resistant regions, particularly rich in C-G pairs. They make up around 15% of all the bands but contain around 65% of all the genes mapped.

Fig 8: T banding

Banding techniques using other stains:

2. Q-Banding:

Quinacrine mustard is an alkylating agent which fluoresces brightly under UV light. These bands are visible under a fluorescence microscope. The alternating bands of bright and dull fluorescence are called Q bands. The bright bands are AT rich region and the dull bands are GC rich region (similar to G banding). Q bands are useful in distinguishing the human Y chromosome and various chromosome polymorphisms involving satellites and centromeres of specific chromosomes.

Fig 9: Q-banding

NOR banding

NOR banding involves the staining of “nucleolar organizing region” by silver stain (silver nitrate solution).The NOR contains rRNA genes. It is thought that the silver nitrate attaches to the nucleolar proteins and not the rDNA in itself. In humans, the NORs are found on the short arms of the chromosomes 13, 14, 15, 21 and 22, the genes RNR1, RNR2, RNR3, RNR4, and RNR5 respectively. They code for 5.8S, 18S, and 28S rRNA. The silver stain usually stains the transcriptionally active rRNA genes. Changes in the NOR number and size help explain changes in transcriptional activity in different environment and conditions.

Fig 10: Stained NORs

(Just for info: Read how banding technique allows to find diversification in the species of fishes

DAPI/Distamycin A Staining:

This is a fluorescent staining technique for labelling a specific subset of C bands. DAPI/Distamycin A staining is used in identification of peri-centromeric breakpoints in chromosomal rearrangements and chromosomes that are too small for standard banding techniques.

4′-6-diamidino-2-phenylindole (DAPI) is a DNA-binding AT-specific fluorochrome. which gives blue fluorocence. Distamycin A is an DNA-binding AT-specific oligopeptide antibiotic. Distamycin A pretreatment results in decrease in fluorescence by DAPI staining, allowing only some specific regions of constitutive heterochromatin to brightly fluoresce.

Fig 11: Comparison between only DAPI and DAPI/Distamycin A staining.

These bright bands include the constrictions of chromosomes 1, 9 and 16, the short arm of chromosome 15, and the distal part of the Y.

Hence different banding technique help in staining certain regions darkly or brightly . These regions can be compared with that in homologous chromosomes or chromosomes of different individuals or species to obtain information on diseases, evolution and parentage.

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Also read other posts by The Biotech Notes:

ELISA

Biofilms: Establishing the colony..

PCR: What is that?

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For Students preparing for CSIR-JRF-NET (Life Science, India), here are few books with good reviews.

UGC-CSIR NET/JRF Common Paper-I 15 Years Solved Papers (With Explanation)

Fundamentals of Life Sciences Vol – 1

CSIR-JRF-NET: Life Sciences Fundamentals And Practice Part 1 & Part 2 Combo

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Books on cytogenetics

Fundamentals of Cytogenetics and Genetics (by Mahabal Ram)

Cytogenetics (by P.K. Gupta)

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(Image sources: Fig 8: T banding; Fig 9: Q banding; Fig 10: Stained NORs; Fig 11: DAPI (Thermofisher) and DAPI/Distamycin A staining)