CHROMOSOME BANDING TECHNIQUES INTRODUCTION:A chromoso chromosome me banding banding pattern pattern is compris comprised ed of alterna alternating ting light light and dark stripes, stripes, or bands that appear along its length after being stained with a dye. OR The treatment of chromosomes of chromosomes to reveal characteristic patterns of horizontal bands like bar codes is known as chromosomal banding
A unique banding pattern is used to identify identify each chromosome chromosome and to diagnose diagnose chromosomal chromosomal aberrations, including chromosome breakage, loss, duplication or inverted segments.
History of Chromosome Banding Techniques
Stain or Banding Technique
Investigator
Year
Q-banding
Caspersson, Zech, Johansson
1970
G-banding (by trypsin)
Seabright
1971
G-banding (by acetic-saline)
Sumner, Evans, Buckland
1971
C-banding
Arrighi, Hsu
1971
R-banding (by heat and Giemsa)
Dutrillaux, Lejeune
1971
G-11 stain
Bobrow, Madan, Pearson
1972
Antibody bands
Dev, et al
1972
R-banding (by fluorescence)
Bobrow, Madan
1973
Shafer
1973
T-banding
Dutrillaux
1973
Replication banding
Latt
1973
Silver (NOR) stain
Howell, Denton, Diamond
1973
High resolution banding
Yunis
1975
DAPI/distamycin A stain
Schweizer, Ambros, Andrle
1978
Restriction endonuclease banding
Sahasrabuddhe, Pathak, Hsu
1978
In vitro
bands (by actinomycin D)
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Chromosomal Banding Patterns
•
Most of chromosomes, at the prophase and the metaphase, are characterized by a banding pattern. But this banding pattern is more evident and clear in the case of larger chromosomes such as the polytene chromosomes of drosophila melanogaster, or the fruit fly.
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The banding patterns are the regions rich in heterochromatin, where the histone -DNA interaction is more. These complexes can be stained very easily by the conventional nuclear dyes or chromosomal dyes such as orcein.
•
The regions between the bands are actually the active regions of the chromatin where more genes are present, but the quanti quantity ty of DNA is very low and therefore therefore the histone proteins. That is why they appear unstained or colored lightly. These interband regions can be detected with immuno-staining by using fluorescently labeled antibodies against the DNA-dependent RNA polymerase, which is usually seen with euchromatin regions required for the process of transcription.
•
Specialized staining techniques are now available, which enable one to differentiate or precisely identify individual chromosome homologes, chromosome regions, and/ or chromosome bands. A renewed interest in the chromosomal or cytogenetic status of various species has been generated by the advancements of genetic mapping techniques utilizing fluorescence in situ hybridization or FISH.
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Depending upon the type of dye or fluorochrome or the chromosome pretreatment, there can be different types of banding patterns. They include banding patterns such as -banding etc. G-banding, Q-banding, C-banding, and R -banding
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The data data generat generated ed by multip multiple le chromo chromosom somee banding banding techni techniques ques can be used used for karyotypic analysis.
•
At a conference in Paris in 1971 , the bands visualized in human mid- metaphase karyotypes by such techniques were assigned a nomenclature in which letters p &
q
represent respectively, the short & long “arms” of a metaphase chromatid ; these arms are then subdivided by numbers.
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•
ISCN is International System For Human Cytogenetic Nomenclature . Each area of
chromo chromosom somee is given given number number,, lowest lowest number number closes closestt (proxi (proximal mal)) to centro centromer meree &
highest number at tips (distal) to centromere. Sub classifications of banding methods ISCN 1985:
Three letter code to describe banding techniques first letter – type of banding second letter – general technique Third letter – the stain e.g. , QFQ – Q bands by florescence using quinacrine
DIFFERENT CHROMOSOME BANDING TECHNIQUES 1. Q-banding: •
Quin Quinac acri rine ne must ustard, ard, an alky alkyllati ating agen agentt, was was the the first rst chem chemic ical al to be used used for chromosome banding. T. Casperss and his his coll collea eagu gues es,, who devel develope oped d the the Caspersson on and technique,
noticed
that
bright
and
dull
fluorescent
bands
appeared
after chromosomes stained with quinacrine mustard were viewed under a fluorescence microscope. •
Quinacrine dihydrochloride was subsequently substituted for quinacrine mustard.
•
The alternating bands of bright and dull fluorescence were called Q bands. Quinacrine bright bands were composed primarily of DNA that was rich in the bases adenine (A) and thymine (T), and quinacrine-dull bands were composed of DNA that was rich in the bases guanine (G) and cytosine (C).
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•
This banding pattern is obtained by treating with a fluorochrome or the fluorescent dye quinacrine. They can be identified by a yellow fluorescence of different intensity.
•
Most parts of the stained DNA are heterochromatin.
•
A-T regions are seen more in heterochromatin than in euchromatin. Therefore, by this banding method heterochromatin regions are labeled preferentially.
•
The characters of the banding regions and the specificity of the fluorochrome are not exclusively dependent on their affinity to regions rich in A- T, but it depends on the distribution of A- T and its association with other molecules such as histone proteins.
Advantages:
It is a simple and versatile technique,
It is used where G – band is not acceptable. It is used as a method of identifying chromosomes 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.
2. G-banding: •
Giemsa has become the most commonly used stain in cytogenetic analysis. Staining a metaphase chromosome with a Giemsa stain is referred to as G-banding. This technique is not a fluorochrome -based pretreatment.
•
It is well suited to animal cells. During mitosis, the 23 pairs of human chromosomes condense and are visible v isible with a light microscope.
•
A karyot karyotype ype analys analysis is usuall usually y involv involves es blocki blocking ng cells cells in mitosi mitosiss and staini staining ng the condensed chromosomes with Giemsa dye.
•
The dye stains regions of chromosomes that are rich in the base pairs Adenine (A) and Thymine (T) producing a dark band. The regions of the chromosome that are rich in guanine and cytosine have little affinity for the dye and remain light.
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•
Unlike
Q-banding,
most
G-banding
techniques
require
pretreating
the chromosomes with either salt or a proteolytic (protein-digesting) enzyme. •
"GTG banding" refers to the process in which G-banding is preceded by treating chromosomes with trypsin.
•
A common misconception is that bands represent single genes, but in fact the thinnest bands contain over a million base pairs and potentially hundreds of genes. For example, the size of one small band is about equal to the entire genetic information for one bacterium.
•
Standard G-band staining techniques allow between 400 and 600 bands to be seen on metaphase chromosomes.
G-banded metaphase from a normal female. •
With high-resoluti high-resolution on G-banding G-banding techniques, techniques, as many as two thousand different different bands have been catalogued on the twenty-four human chromosomes.
•
Jorge Yunis introduced a technique to synchronize cells so they are held at the same stage in the cell cycle. Cells are synchronized by making them deficient in folate, thereby inhibiting DNA synthesis. By rescuing the cells with thymidine, DNA synthesis is initiated and the timing of the prophase and prometaphase stages of the cell cycle can be
predicted.
Yunis's
technique
allows
more
bands
to
be
resolved,
as chromosomes produced from either prophase or prometaphase are less condensed and are thus longer than metaphase chromosomes.
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Applications:
Most widely used principle methods for demonstrating euchromatic bands than other two. 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 The
inef ineffe fect ctiv iven enes esss
of
dete determ rmin inin ing g
smal smalll
tran transl sloc ocat atio ions ns,,
dete detect ctin ing g
microd microdele eleti tions ons & charac character terizi izing ng the chromo chromosom somes es of cell cell lines lines which which are complex.
3. C-banding: •
The name C-banding originated originated from centromeric centromeric or constituti constitutive ve heterochrom heterochromatin. atin. C banding stains areas of heterochromatin, which is tightly packed and repetitive DNA.
•
The centromere appears as a stained band compared to other regions.
•
The techni technique que involv involves es a pretre pretreatm atment ent with with alkali alkali before before staini staining. ng. The alkali alkaline ne pretreatment leads to the complete depurination of the DNA. The remaining DNA is again again renatu renatured red and staine stained d with with Giemsa Giemsa soluti solution on consist consisting ing of methyl methylene ene azure, azure, methylene violet, methylene blue, and eosin.
•
In this staining the heterochromatin take a lot of stain but the rest of the chromosomes stain only a little.
Applications:
This This band bandin ing g techn techniq ique ue is well well suit suited ed for for the the chara charact cter eriz izat atio ion n of plant plant chromosomes.
7 C banding banding is valuabl valuablee for the identi identific ficati ation on of chromo chromosom somee partic particula ularly rly in insects of plants Useful in the identification of meiotic chromosomes even in the species such as mammals which shows good G banding pattern on mitotic chromosome. C banding is valuable to identify bivalents at diakinesis using both centromere positions. C bands used for paternity testing and gene mapping. 4. R-banding: •
This is known as a reverse banding technique . This technique results in the staining of areas rich in G-C that is typical for euchromatins.
•
R-banding involves pretreating cells with a hot salt solution that denatures DNA that is rich in adenine and thymine. The chromosomes are then stained with Giemsa.
•
R-banding is helpful helpful for analyzing analyzing the structure structure of chromosome chromosome ends, Advantage - R-banding since these areas usually stain light with G-banding.
•
G-, Q-, and R-bandings are not observed with plant chromosomes.
5. Hy-banding: •
This is a common technique used with plant cells.
•
The technique involves a pretreatment of the cells in which the cells are warmed in the presence of HCl and then stained with acetocarmine.
•
The pattern of Hy-band is different from that of C-bands. The binding of histone protein to DNA and its complete extraction has an impact on the binding ability of acetocarmine and formation of bands.
6. NOR-staining: (Silver Nucleolar Organizing Region Staining)
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•
NOR is an abbreviation for "nucleolar organizing region," refers to a silver staining method that identifies genes for ribosomal RNA that were active in a previous cell cycle.
•
Chromosomes are treated with silver nitrate solution which binds to the Nucleolar Organizing Organizing Regions Regions (NOR), (NOR), i.e., the secondary secondary constricti constrictions ons (stalks) (stalks) of acrocentri acrocentricc chromosomes.
7. DAPI/Distamycin A Staining •
The DAPI/ DAPI/dis distam tamyci ycinn- A fluor fluoresc escent ent staini staining ng techni technique que was first first descri described bed by Schweizer, Schweizer, Ambros, and Andrle as a method method for labeling labeling a specific specific subset of C bands. (Gustashaw, 1991).
•
The combination of the fluorescent dye, DAPI (4, 6-Diamidino-2-Phenylindole) with a non-fluorescent counterstain, such as Distamycin A, will also stain DNA that is rich in adenine and thymine.
•
It will particularly highlight regions that are on the Y chromosome, on chromosomes 9 and 16, and on the proximal short arms of the chromosome 15 homologues , or pair.
•
The DAPI/Distamycin A staining technique is useful in identifying pericentromeric breakpoints in chromosomal rearrangements and in identifying chromosomes that are too small for standard banding techniques.
9. T-Banding: •
T-bandi T-banding ng is used used to stain stain the telome telomeric ric region regionss of chromo chromosom somes es for cytogen cytogeneti eticc analysis.
•
Telomeric Telomeric (or terminal) terminal) banding was first first reported reported by Dutrillaux Dutrillaux,, who used two types of controlled thermal denaturation followed by staining with either Giemsa or acridine orange.
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•
The T bands apparently represent a subset of the R bands because they are smaller that the corresponding R bands and are more strictly telomeric. (Gustashaw, 1991).
