CHROMOSOMAL BANDING TECHNIQUES

What are chromosomes…? Thread like structures found in the nucleus of a cell. They store and carry DNA They are carriers of GENE (SUTTON & BOVERY- 1902) They help in the transmission of heredity (MORGAN-1933)

What are bands..? Part of chromosome, clearly distinguishable from its adjacent segments by appearing darker with banding techniques. Produce landmarks along the length of metaphase chromosomes. Allow recognition of individual chromosome within a genome & identification of specific segments of individual chromosomes.

Chromosomal banding techniques Falls into 2 principle groups: those resulting in bands distributed along the length of the whole chromosomes Eg: G,Q & R bands those that stain a restricted number of specific bands or structures. Eg: C & NOR bands

Causes of banding Occurrence of repetitive DNA. Difference in base composition of DNA. Difference in protein component. Difference in the degree of packing of DNA or DNA complex.

Classification of bands Based on the techniques used: Constant heterochromatic bands Fluctuant bands

Constant heterochromatic bands Visible in interphase & throughout division. Constant in size. Equivalent to C, GII & n bands. Associated with highly repetitive DNA. Eg: bands in plants, insect, amphibia..

Fluctuant bands Not distinguishable in interphase. Vary in size throughout prophase. Equivalent to Q,G, & R bands. Appear to be centers of mitotic chromosome condensation. Eg: higher vertebrates.

Principle of banding Based on staining of chromosome with a dye. Single strands of RNA or DNA are able to pair with their complementary base sequences.

Objectives For obtaining and understanding chromosome structure. Understanding the behaviours of chromosomes in terms of their substructures. Improving the reliability of banding techniques.

Uses For karyotyping, identifying abnormalities of chromosome number, translocation, deletion, inversion or amplification of chromosomes. Predicting evolutionary relationships between species.

Conventional staining enables measuring of chromosome length, centromeric position and arm ratio. Allow clear differentiation of chromosome segments. Favours identification of chromosome segments with specific molecular complexity.

Chromosome banding techniques G banding By Giemsa stain. For staining metaphase chromosomes. Chromosomes are pretreated with salt or proteolytic enzyme. Stains region of DNA rich in adenine and thymine.

Regions with guanine and cytosine have little affinity so remain light. Standard G banding allow 400-600 bands on metaphase chromosomes.

R banding Reverse pattern of G bands. Pretreating cells with hot salt solution causing denaturation of DNA, rich in adenine and thymine. Stained with Giesma stain. For analyzing the structure of chromosome ends.

C banding Staining heterochromatin area, which is tightly packed and repetitive DNA. Useful in humans for staining centromeric chromosome regions. Secondary constrictions of human chromosomes 1,9,16 and distal segment of Y chromosome long arm.

Q banding By Quinacrine mustard, an alkylating agent or Quinacrine dihydrochloride. Q bands are alternating bright and full fluorescence bands. Bright bands, regions of DNA rich in adenine and thymine. Dull bands, regions with guanine and cytosine. For identifying human chromosome Y and various polymorphisms.

Cat Q band Q banding

T bands For staining telomeric regions. By Giemsa stain or Acridine orange, after controlled thermal treatment.

Cat telo

O bands Used in plants. No denaturation. Involves the pretreatment of tissues, fixation in acetic acid-ethanol in strong concentration of 1xssc at 27-28C washing, staining in acid Orcein and mounting in 45% acetic acid. Reaction involves the DNA – Protein linkage, since Orcein, is an amphoteric dye stains DNA and protein.

Gradual removal of non – histone proteins are responsible for ‘O’ bands. Allows localization of major and minor repeated sequence in chromosomes.

N or NOR staining For nuclear organizing regions. Represent structural non-histone protein linked to nucleolar organizers in different eukaryotic chromosomes. Silver nitrate solution binds to nucleolar organizing regions (secondary constrictions of acrocentric chromosomes).

Hy banding For plant cells. Applied to somatic chromosomes of members of Liliflorae.

CT bands Banding of centromeric and telomeric segments. Incubating in barium hydroxide and staining in “all stain” (4,5,4’,5-dibenzo 3,3’-diethyl 9-methyl, thiocarbocyanine bromide).

In situ hybridization- ISH Localisation and mapping of different segments of chromosomes and gene loci at microscopic level. Use probe sequence tagged with radioisotopes or fluorescent compounds. Multicolored FISH, for gene mapping and detection of abnormalities.

Alpha satellite FISH FISH locus probe

Applied with total genome probes –GISH. Help in genome identification, their orientation and establishing genomic relationship between species.

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