Chromosome Banding techniques : Chromosome Banding techniques Chromosome Banding Techniques CHROMOSOME BANDING TECHNIQUES DISCLAIMER: Copyright of some of the figures used from internet and different web sites is duly acknowledged. The copyright stands with its original developer. The information has been gathered here for educational purpose and not for any kind of commercial purpose. BAND : BAND Paris conference in 1971 defined band as a part of a chromosome which is clearly distinguishable from its adjacent segments by appearing darker or lighter with various banding methods Slide 3: In 1958 Caspersson et al. published there first paper describing the use of quinacrine mustard to stain chromosome there by ushered in a new era of chromosome banding
The Paris report (1971) was the first attempt to provide nomenclature for chromosome banding in any species and thus its recommendations have been adopted non human species as well. Idiogram : Idiogram ISCN : ISCN International System for Human Cytogenetic Nomenclature
Each area of chromosome given number
Lowest number closest (proximal) to centromere
Highest number at tips (distal) to centromere Nomenclature: p (pepit) arm = short arm; q=long arm.Band numbering proceeds outwards from the centromere e.g., bands p11 (one-one NOT eleven) or p23(p two-three NOT twenty three : Nomenclature: p (pepit) arm = short arm; q=long arm.Band numbering proceeds outwards from the centromere e.g., bands p11 (one-one NOT eleven) or p23(p two-three NOT twenty three Karyotyping : Karyotyping Four main staining methods to identify chromosomes
G banding - Giemsa
Q banding - Quinacrine
R banding - Reverse
C banding - Centromeric (heterochromatin)
Ag-NOR stain - Nucleolar Organizing Regions (active) Sub classifications of banding methods ISCN 1985 : 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
Ex , QFQ – Q bands by florescence using quinacrine G - banding : G - banding G – bands may be defined as a system of dark and light bands throught the length of the euchromatin part of the chromosomes.
A staining technique in which chromosomes are treated with trypsin, then with Giemsa stain. Also called Giemsa chromosome banding stain. Karyotyping – cell preparation : Karyotyping – cell preparation Need metaphases
Culture cells until sufficient mitotic activity
Add colchicine (or colcemid) to arrest in metaphase
prevents mitotic spindle fibres forming
Add hypotonic salt solution to swell cells
Fix with mix of methanol;acetic acid
Want long chromosomes with none overlapping Preparation of G banded karyotype : Preparation of G banded karyotype Normal male karyotype : Normal male karyotype 13, 18, 21 gene poor
Very dark chromosomes
21 smaller than 22
Wrong way around
22 twice as many genes as 21
200 on 21
400 on 22 Normal female karyotype : Normal female karyotype Slide 14: Color enhanced tetraploid ‘African’ chromosomes
Solution: Utilize Giemsa banding techniques and image analysis to identify and karyotype tetraploid alfalfa chromosomes. (See Bauchan and Hossain, 1999; 2001; 2002 & 2003). Slide 15: G- bands can be demonstrated easily and regularly on chromosomes of higher, amnioate, vertebrates ( reptiles, birds and mammals )
G – bands normally not produced in plants Slide 16: chromosomes much more contracted at metaphase than those of mammals.
2) limits of resolution of light microscope, any bands present would be too close to be resolved
3) technical factors
4) amphibian fishes and other eukaryotes studied had much more homogeneous DNA, not seperable into AT rich and GC rich components
Therefore only experiment to find G – bands in higher vertibrates Slide 17: Positive G – bands Negative G – bands
Positive Q – bands negative Q – bands
Negative R – band positive R bands
Early condensation late condensation
Late replicating DNA early replicating DNA
Tissue specific genes house keeping genes
A+T rich region G+C rich region Applications : Applications most widely used principle methods for demonstrating euchromatic bands than other two.
Chromosome abnormalities; aneuploidy, breakage and rearrangement.
Chromosome of cultured cells.
Chromosome banding and cancer.
Homogeneity of staining regions.
High resolution banding ( microcytogenetics) Disadvantages : Disadvantages the ineffectiveness of determining small translocations
characterizing the chromosomes of cell lines which are complex. C – banding method : C – banding method Those method which demonstrate constitutive heterochromatin. A C- band is a unit of chromatin stained by these methods
However, when several completely different methods give similar results on a species that is well known cytogenetically it is reasonable to regard as a C band method
Used to identify centromeres / heterochromatin
contain repetitive sequences
highly condensed chromatin fibres Slide 21: This method usually involves treatment with acid, alkali or elevated temperature.
Cellular DNA is denatured by these treatments
Highly repetitive DNA such as constitutive heterochromatin renatures under prescribed conditions but not unique DNA thereby creating differential staining reaction. Slide 22: BSG ( Barium hydroxide / Saline / Giemsa ) technique can be regarded as a standard C banding method for virtually all plant and animal chromosomes
Mechanism of C banding can be produced by reagents whose actions is essentially on proteins and not on DNA
Proteins in the C bands had a greater affinity for giemsa Applications : Applications C banding is valuable for the identification of chromosome particularly 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 position.
C bands used for paternity testing and gene mapping. Slide 24: C-banded metaphase cell. G – 11 banding : G – 11 banding G – 11 banding - Bobrow et al.
Priciciple – the method consists of simply staining chromosome preparation with a Giemsa solution at a pH about 11
Specific magenta staining of the centromeric heterochromatin of chromosome 9, while everything else was stained blue Applications : Applications study of aneuploidy for chromosome 9 in human spermatozoa
distinction of rodent and primate chromosomes in somatic cell hybrids N – bands : N – bands Developed to show the NOR
Along with C banding mehod, proved much superior method for plants Slide 29: N-banded chromosomes of diploid M. sativa ssp. coerulea. Note the multiple bands and compare to C-banding pattern Q – banding : Q – banding Quinacrine mustard, an alkylating agent, was the first chemical to be used for chromosome banding
T. Caspersson and his colleagues, who developed the 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 Slide 31: 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 and thymine, and quinacrine - dull bands were composed of DNA that was rich in the bases guanine and cytosine.
Binding of quinacrine mustard to DNA by intercalation, with alkylation of guanine a secondary binding mode. Slide 32: The Q banding must be the result of more efficient excitation of fuorescence in some chromosomal region than others.
The main cause of differential excitation of fluorescence is the base composition of the chromosome , and, more specifically, that the fluorescence of quinacrine is brighter when it is bound to AT rich DNA than when it is bound to GC rich DNA..
Quinacrine fluorescence is an indicator of AT richness of the DNA to which it binds Slide 33: Q - Band Advantages : 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 : Disadvantages generally associated associated with any fluorescence technique: impermanence of the preparations
the tendency to fade during examination R – banding : R – banding R-banding is the reverse pattern of G bands
G-positive bands are light with R-banding methods, and vice versa.
. 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. Slide 37: R - Band Chromosome X : Chromosome X Chromosome X : G-banding, diagram and R-banding - Claude Léonard, Jean-Loup Huret Chromosome Y : Chromosome Y Chromosome Y : G-banding, diagram and R-banding - Claude Léonard, Jean-Lo Chromosome 1 : Chromosome 1 Chromosome 1 : G-banding, diagram and R-banding - Claude Léonard, Jean-Loup Huret Advantages : Advantages R-banding is helpful for analyzing the structure of chromosome ends, since these areas usually stain light with G-banding. T – banding : T – banding Terminal banding
It uses, high temperature, pH – 6.7. and giemsa staining
Most efficient banding pattern for chromosome terminal banding
Identification of translocation Slide 43: THANK YOU