Chromosome Banding Techniques

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Presentation Transcript

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 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 : 

Disadvantages the ineffectiveness of determining small translocations detecting microdeletions. 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 Heterochromatic regions 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