Chromosome Banding

and Identification
HUMAN CHROMOSOMES
3 BASIC CATEGORIES
Metacentric chromosome with its centromere in the middle
Sub-metacentric one with the centromere closer to one end
Acrocentric one with the centromere almost at one end
Karyotype
• Based on decreasing relative size and
centromere position
• Comprised of seven groups labeled A
through G was devised
• X chromosome belonged to the third
or “C” group, whereas the Y was often
placed separately
Chromosomal Banding
alternating dark and light stained demarcations that appear along
the length of each chromosome
treatmentof chromosomesto reveal characteristic patterns of
horizontal bands like bar codes
specific for each chromosome pair
enables the identification not only of individual chromosomes but
also of regions within each chromosome
Methods
• Giemsa or G-banding
• Quinacrine mustard or Q-
banding
• Reverse or R-banding
• Constitutive heterochromatin
or C-banding
Q-banding
• Quinacrine mustard alkylating agent = yellow fluorescence
• Quinacrine dihydrochloride was subsequently substituted for
quinacrine mustard.
• Bright and dull fluorescence  Q bands
• Bright bands composed primarily of DNA rich in adenine (A) and
thymine (T))
• Dull bands composed of DNA rich in basesguanine (G) and cytosine
(C)
Q-banding
Advantages:
• It is a simple and versatile technique,
• It is used where G – band is not acceptable. It is used as a method of
identifyingchromosomes in combination with other procedure
• Study of heteromorphism
• Study of human Y chromosome
Disadvantages:
• Generally associated with any fluorescence technique: impermanence of
the preparations, the tendency to fade during examination
G-banding
• Giemsa most commonly used stain
• Not a fluorochrome -based pretreatment.
• It is well suited to animal cells. During mitosis, the 23 pairs of human
chromosomescondense and are visible with a light microscope.
• Stains regions of chromosomes rich in Adenine (A) and Thymine (T) producing a
dark band.
• Guanine and cytosine have little affinity for the dye and remain light.
• Require pretreatingthe chromosomes with either salt or a proteolytic (protein-
digesting) enzyme.
• "GTG banding"  G-banding is preceded by treating chromosomes with trypsin
G-banding
Applications:
• Most widely used principle methods for demonstrating euchromatic bands.
Chromosome identification, Chromosome abnormalities; aneuploidy,
breakage and rearrangement, Chromosome of cultured cells, Chromosome
banding and cancer,Homogeneity of staining regions, Gene mapping& High
resolution banding (microcytogenetics)
Disadvantages:
• The ineffectiveness of determining small translocations, detecting
microdeletions & characterizing the chromosomes of cell lines which are
complex
matin take a lot of stain but the
onlyC-banding
a little.
• Centromeric or constitutive heterochromatin
• stains areas of heterochromatin, which is tightly packed and repetitive

suited for the characterization 
DNA.
• Centromere appears as a stained band compared to other regions.
• Pretreatment with alkali before staining with Giemsa solution consisti
ng of methelene azure, methylene violet, methylene blue, and eosin.

entification of chromosome par
C-banding
Applications:
• Well suited for the characterization of plantchromosomes
• Identification of meiotic chromosomes even in the species such as mammals
which shows good G banding pattern on mitotic chromosome.
• Identify bivalents at diakinesis using both centromere positions.
• Used for paternity testing and gene mapping
R-banding
• Reverse banding technique
• Staining of areas rich in G-C that is typical for euchromatins
• Pretreating cells with a hot salt solution that denatures DNA that is
rich in adenine and thymine  stained with Giemsa

Advantage
• Helpful for analyzing the structure of chromosome ends, since these areas
usually stain light with G-banding.
 Hy-banding
• Common technique used with plant cells.
• Pretreatment of the cells in which the cells are warmed in
the presence of HCl and then stained with acetocarmine.
• Binding of histone protein to DNA and its complete extraction has an
impact on the binding ability of acetocarmine and formation of bands
NOR-staining: (Silver Nucleolar
Organizing Region Staining)
• “Nucleolar organizing region"
• Silver staining method that identifies genes for ribosomal RNA that
were active in a previous cell cycle
• Treated with silver nitrate solution which binds to the
Nucleolar Organizing Regions (NOR),
secondary constrictions (stalks) of acrocentric chromosomes
DAPI/Distamycin A Staining
• First described byas a method for labeling a specific subset of C bands
• The combination of the fluorescent dye, DAPI (4, 6-Diamidino-2-
Phenylindole) with anon-fluorescent counterstain, such as Distamycin
A, will also stain DNA that is rich inadenine and thymine
• Particularly highlight regions that are on the Y chromosome, on
chromosomes 9and 16, and on the proximal short arms of the
chromosome 15 Homologues , or pair.
• Identifying pericentromeric breakpoints in chromosomal
rearrangements and in identifying chromosomes that are too small
for standard banding techniques
T-Banding
• Stain the telomeric regions of chromosomes for cytogenetic analysis
• Used two typesof controlled thermal denaturation followed by
staining with either Giemsa or acridineorange
• Represent a subset of the R bands because they are smaller that the
corresponding R bands and are more strictly telomeric