SEMINAR REPORT
CHROMOSOMAL BANDING
By:
Department Of Biotechnology
Administrative Management College.
Bangalore University, Bangalore.
Karnataka.
2001-2002.
I wish to express my thanks to Dr. Sabitri M. Bhat, Lecturer, Department of Biotechnology, A.M.C. College for her valuable help in clearing my doubts on the topic.
I wish my humble thanks to Dr. D.V.S.S.R. Prakash, Head, Department of Biotechnology, A.M.C. College for providing me all the necessary facilities for collection of data by visiting libraries of IISc and GKVK, Bangalore.
M. Sc. I year ( 2001-02)
Department of Biotechnology
Chapter - I : INTRODUCTION
Chapter -2 : HISTORY OF CHROMOSOMAL BONDING
Chapter-3 : IMPROVEMENT OF TECHNIQUES FOR
STUDY OF CHROMOSOME STRUCTURES
Chapter - 4 : CLASSIFICATION AND COMPOSITION OF
BANDS
Chapter - 5 : TYPES OF BANDING TECHNIQUES
Sub-topics 5(A) : CONVENTIONAL STAINING
Sub-topics 5(B) : FLUORESCENT BANDING OR Q-BANDING.
Sub-topics 5(C) : G-BANDING
Sub-topics 5(D) : C-BANDING
Sub-topics 5(E) : R-BANDING
Sub-topics 5(F) : T-BANDING
Sub-topics 5(G) : O-BANDING
Sub-topics 5(H) : N-BANDING OR NOR-STAINING
Sub-topics 5(I) : HY-BANDING
Sub-topics 5(J) : RESTRICTION ENZYME BANDING
Chapter - 6 : CONCLUSION
Chapter - 7 : REFERENCES
Chromosome identification has traditionally been dependent on their morphological characteristics such as relative lengths, arm ratio and presence or absence of secondary constrictions. Then the differential banding patterns of chromosomes, usually observed at specific regions on particular levels, were initially developed for the analysis of human chromosome segments. These bands are made visible through low and high intensity regions under the fluorescence microscope or as differentially stained areas under the light microscope (see Lubs et al., 1973; Houghton, 1974; Miller, Miller and Warburton, 1974; Sharma, A. and Talukder, 1974; Yunis, 1974; Vagner-Capodano, Noel and Stahl, 1975; Borgaonkar, 1976;Pearson and Van Egmond-Cowan, 1976; Schwarzacher,1976; proceedings of Leiden and Helsinki Chromosome Conferences, 1974 and 1977; Szabo and Papp, 1977 for review). These methods were then extended first to different animals and later to plant chromosomes.
The advent of molecular hybridization, which revealed the functionally different segments in a eukaryotic chromosome, led to the banding pattern technique. With the development of chromosome banding techniques how ever, the development of chromosome banding techniques during the last more than two decades provided a very useful additional tool for identification of individual chromosomes within the complement. These techniques, not only allow the identification of chromosomes that differ morphologically with greater degree of confidence, but also allow identification of chromosomes that possess similar morphological attributes. It also permits us to establish a correlation between linkage maps and cytological maps.
The banding techniques are based on the identification of chromosome segments that predominantly consist of either GC or AT rich regions or of constitutive heterochromatin. There is also general agreement that the techniques, which involves denaturation of DNA followed by slow renaturation, permits identification of constitutive heterochromatin, because it mainly consists of repetitive DNA. A variety of different kinds of bands (e.g., Q, C, G or R bands) have been studied in animal materials including humans, mouse, pig, domestic mosquito, etc. and the results have been found to be consistent. Heteromorphy for chromosome bands has also been demonstrated in many established cases, so that the possibility of using bands as ‘markers’ has been established in some cases.
It is based on the principle that single strands of RNA or DNA are able to recognize and pair with their complementary base sequences. A denatured DNA duplex, on renaturation, undergoes pairing at complementary sequences. Highly repeated sequences show rapid rate of reannealing. The reassociation kinetics gives an indication of sequence complexity of DNA.
There are three related objectives in studying mechanism of chromosome banding obtaining and understanding of chromosome structure, understanding the behaviors of chromosome in terms of their substructure; and improving the reliability of banding techniques.
Similarities in organism are commonly interpreted as the result of common ancestry. Since chromosomes are the carriers of heredity, similarities in chromosomes could have special significance in studying the ancestry and relationships of species. Many studies comparing chromosome banding have been conducted.
CAUSES OF BANDING:
Various causes have been ascribed to the occurrence of the chromosome bands. Of them four important factors are:
1) The occurrence of repetetive DNA.
2) Differences in the base composition of DNA,
3) Difference in the protein component
4) Difference in the degree of packing of DNA or DNP-complex.
(Britten and Khone, 1971; Walker, 1971; Comings, 1972; Pardua and Gall, 1972; Schwarzacher, 1976 ).
|
Stain or Banding Technique |
Investigator |
Year |
|
Q-banding G-banding (by trypsin) G-banding (by acetic-saline) C-banding R-banding (by heat and Giemsa) G-11 stain Antibody bands R-banding (by fluorescence) In vitro bands (by actinomycin D) T-banding Replication banding Silver ( NOR) Stain High resolution banding DAPI/distamycin A stain Restriction endonuclease banding |
Caspersson, Zech, Johansson Seabright Sumner, Evans, Buckland Arrighi, Hsu Dutrillaux, Lejeune Bobrow, Madan, Pearson Dev. et al Bobrow, Madan Shafer Dutrillaux Latt Howell, Denton, Diamond Yunis Schweizer, Ambros, Andrle Sahasrabuddhe, Pathak, Hsu |
1970 1971 1971 1971 1971 1972 1972 1973 1973 1973 1973 1973 1975 1978 1978
|
IMPROVEMENT OF TECHNIQUES FOR STUDY OF CHROMOSOME STRUCTURES.
To analyze certain types of chromosome aberrations of clinical importance the primary requisites are the recognition and identification of the individual chromosome region to be studied. These steps are absolutely essential to study their type of work, as several different methods have to be applied one after the other on the same chromosome region, and the results of the analyses in different metaphase plates have to be compared.
In most cases the identification presents great technical difficulties. Chromosome size and morphology differ very much in different animal and plant species. In most objects of interest, however, the size is rather close to the resolution limit of the optical microscope, and the chromosomes carry few morphological makers. Thus, very often, it is not even possible to identify whole chromosomes with conventional cytological techniques. This is the case for most of the mammals, and is especially true for man, in whom only 7 of the 24 chromosome types can be recognized by conventional staining techniques.
The rapid development of mammalian cytogenetics, including clinically applied human cytogenetics, in the last decade has emphasized the need to develop better chromosome recognition procedures. At present the larger part of the general bio-medical, cell biological work is centered around man and a small number of higher mammals. It is logical, therefore, in seeking improved chromosome region identification techniques, to concentrate on these species, in spite of the obvious difficulties involved.
Quantitative cytochemical chromosome work started on the late thirties when the first Ultramicrospectrophotometers were developed. The main problem then was the relation between the protein and nucleic acid metabolisms (Caspersson, 1950). During the following two decades, quantitative cytochemical mainly interphase cells. In the early sixties, biophysics group Medical Noble Institute, Sweden with Gosta Lomakka carried a programme for the development of ultramicrospectrophotometer, ultramicrointerferometer, and micro-X-ray- absorbometric techniques to the very limit of resolution in order to get chemical information from chromosome parts and other nuclear structures. It proved possible for all techniques to get very close to the limit of resolution of the light optical microscope (Caspersson and Lomakka, 1970).
With the most refined Ultramicrospectrophotometric procedures the chromosome classification could be done in some objects. This technique was useless in practical work for some organisms e.g., man and the mouse, where the difference between many chromosomes were too small.
One step forward was to determine the DNA-distribution pattern along the chromosome by scanning the chromosome cross-wise with a very small measuring aperture moved in a very tight scanning pattern (Carlson et al. 1963; Heneen and Caspersson 1973; Caspersson et al. 1970b)
The first promising indication by this method came from rye, with seven chromosome pairs of a size comparable with those met in the human karyotype. On measuring the DNA distribution pattern Caspersson along with the cytologist Waheeb Heneen saw that the patterns were so characteristics that they could serve for identification purposes. The defect of this system was that it worked fine for the larger chromosomes in the karyotype, but was not very reliable for the smaller chromosomes. In addition, measurement was extremely difficult and the instrumentation so expensive that the technique could hardly be of any practical value.
With the view to develop much more convenient methods they (Caspersson et. al) started working on fluorescence techniques. These have several great advantages over absorbemetic procedures. One is their extreme sensitively. Another fact is that the discriminative power of a fluorescent microscope for particles adjacent to one another is almost twice that of conventional bright field microscopy in transmitted light (due to the difference between “Selbstlenchter” and “Nicht – Selbstlenchter” according to Abbe). Furthermore, the determination of the amounts of fluorescing substances in an irregular object is obviously much simpler than absorbometric quantitative work.
For fluorescence technique, perfect fluorescing substance has to be choosen. The choosing was done on the basis of fluorescing substances binding to the DNA. At first base binding substances were tried. Then phosphate binding and also carbohydrate- reacting dyes (Feulgen) were tried which showed very few details in metaphase chromosomes searching for these properties led to the choice of certain acridine compounds in the first instance quinacrine mustard (QM) and quinacrine (Q), of which the former gave best results. This compounds bind to DNA, and then the pattern of fluorescence is influenced by the DNA distribution pattern. There are other factors such as presence of proteins which influence the fluorescence patterns by blocking the binding capacity of the DNA in some chromosome regions, and include enhanchment or diminishing of the acridine fluorescence by adjacent DNA base pairs (AT & GC, respt.), (Rigler, 1969; Pachmann and Rigler, 1972; Weissblum and de Haseth, 1972)
Several technical problems arise in ultramicrofluorometry. Firstly, very high-class optics have to be used in order to catch the fine details of the patterns. Secondly, the exposure of the object to the exciting radiation must be as low as possible because of fluorescence fading, which runs relatively fast in these very minute structures. Thirdly, great precision is required both to define the place in the preparation where measurement has to be done, and to set the size and the shape of the area to be measured.
In order to assemble rapidly a large number and range of fluorescence patterns, a less precise but very much faster fluorometer system (Caspersson et al. 1970d) was devised. It was based on microdensitometry of fluorescence photomicrographs of Q-or QM-stained metaphase plates. In it, both negatives and positives can be used; the latter, with the aid of a reflectometer arrangement. The advantage of positives is that one can easily cut them up and make a preliminary sorting of the chromosome pictures to be measured. In routine work, aiming at studies of large numbers of chromosomes, this saves much time. It is now possible to identify all human chromosomes accurately. (Caspersson et al. 1970, 1971, 1972; and Caspersson and Zech, 1972). The current, internationally accepted numbering and defination of human chromosomes are based on these measurements. For statistical studies, the pattern measurements are necessary. In the analysis of chromosome aberrations, fluorometry is a great advantage.
In the figure-2 the typical patterns of the whole human karyotype is given which is the accepted norm for chromosome numbering at present. The principle of numbering the chromosome is, that the shorter the chromosome the higher the number. In addition the numbers were kept that had earlier been assigned to 13 of the 24 chromosome types by means of morphology and autoradiography.
In order to study the statistical significance of the patterns, computerized procedures were used (Caspersson et al. 1972; 1971b; Moller et al. 1972). The first effort concerned the C-group, the 8 chromosomes of which have practically the same length and are indistinguishable from each other by earlier techniques.
In order to assemble a large amount of data, the fluorescence pattern-recording device was equipped with a digital output and a tape punch. It was thus possible to work quite rapidly, especially as the tape could be read directly by the computer. The accuracy of this computerized pattern identification is close to 90%, but can be improved considerably.
Caspersson in collaboration with Dr. Castleman at the Jet Propulsion Laboratory (JPL), Pasadena, developed an entirely automated chromosome recognition system using the automatic microscope and film scanner, connected to a digital computer. Apart from the possibility of recognizing individual chromosomes, the pattern recognition technique also offers opportunities to identify individual chromosome regions. This is of special significance in a) mutagenesis work, b) the study of medically important aberrations, and c) cytochemical chromosome analysis with different kinds of methods. Fluorescence techniques are especially suitable for this purpose because quantitative recordings of pattern details can be made easily and analyzed mathematically by computer.
Further development is done by application of procedures based on TV techniques, which offers another possibility to enhance contrasts. Caspersson et al., 1970; have built up a system in which a TV camera is trained on the negative of fluorescence photograph and the TV picture can be observed in an arbitrarily chosen optical enlargement on a monitor. Contrast enhancement can be obtained electronically in any region of intensity, which means that, regardless of the intensity of the fluorescence of the individual chromosome one can rapidly examine its pattern in minute detail. The camera can also be fitted to a microscope. This means a great increase in speed and convenience in the analysis of metaphase plates for aberrations; the instrument is equipped with an oscilloscope, which simultaneously gives the course of intensity along a freely adjustable scanning line.
The apparatus has been developed further, especially with a view to the important practical problem of identifying very small chromosome aberrations. This has been accomplished by the introduction of a second TV camera whose picture can be relayed to the same monitor as that of that first camera. In this way pictures of two chromosome, e.g. members of one and the same pair, can be juxtaposed with optional enlargements, thus greatly increasing the possibilities of identifying small aberrations in the patterns.
CLASSIFICATION AND COMPOSITION OF BANDS
What is a band?
A band is a unit of chromosome replication and its DNA is relatively homogeneous in base composition. It is a cluster of about 7-25 contiguous replicons, which simultaneously initiate and terminate DNA synthesis during a fraction of the DNA synthesis phase, S-phase, of the cell cycle (Hand, 1978; Lau and Arrighi, 1981; Hameister and Sperling, 1984).
Band reflects a compartmentalization of the mammalian genome and at least eight different compartment classes can be identified by their different base compositions. The GC-richest compartment classes correspond to early replicating DNA (R-band DNA), while the AT-richest compartment classes correspond to late replicating DNA (G-band DNA). A gene’s codon usage is determined mainly by its band location and not by its function.
CLASSIFICATION OF BANDS (A.T. Sumner, 1976).
Bands are usually classified according to the technique used to produce them, so that the same band may be designated differently according to the technique used (e.g. G,Q and R bands), and fundamentally different bands may be given the same designation (e.g. C bands also revealed by G banding). In fact, two distinct classes of bands, and probably three, can be recognized.
1. Constant heterochromatic bands, visible in interphase and throughout division and relatively constant in size. They therefore conform to classical definitions of heterochromatin. These bands are equivalent to C, G11 and N bands, and certain bands revealed by G, Q and R banding techniques. These techniques do not necessarily show all the heterochromatic regions in chromosomes. For example B chromosomes in the grasshopper Myrmeleotettix are entirely heterochromatic, but show distinctive patterns of C-banding, rather than being uniformly dark. Facultative heterochromatin, such as the inactive X chromosome in female mammals, is not normally demonstrated either, although techniques for who showing it have been devised.
All bands in plants, and possibly in some insects and amphibia, appear to be of this type.
2. Fluctuant bands, not distinguishable in interphase, and varying in size throughout prophase until at metaphase they occupy the whole chromosome. This is a genuine fusion of bands, and not merely the result of chromosome contraction bringing them so close together that they can no longer be resolved. The much higher resolution of the electron microscope does not reveal any more bands in contracted chromosomes. These bands are equivalent to those G,Q and R bands, which are not also demonstrated by C-banding, and appear to occur mainly in higher vertebrates, although present in some insects. T bands probably come in this category, but their status is far from certain.
3. Kinetochores.
Eiberg’s Cd- banding technique reveals pairs of dots at the centromers, which can also be shown as objects resistant to alkaline treatment. Since the only known paired structures of this size at the centromers are the kinetochores, the conclusion must be that the Cd bands are the kinetochores
Constant heterochromatic bands: Constant bands are associated with highly repetitive DNA, as satellite DNA, as seen in the mouse. In most species, it is not yet known whether constant bands are associated with particular types of DNA.
No special type of protein has been identified in constant bands. There is a confusion regarding the presence of proteins in constant bands.
Fluctuant bands:
Fluctuant bands change in size throughout mitotic prophase. Fluctuant bands cannot represent particular types of DNA, but are equivalent to meiotic chromomeres and appear to be centers of mitotic chromosome condensation. The differentiation of fluctuant bands must clearly lie with chromosomal proteins.
TYPES OF BANDING TECHNIQUES
There are three conventional staining techniques which are used for chromosome staining. They are by using.
1. Conventional giemsa stain
2. Conventional giemsa stain
3. Leishman’s stain.
The types of banding techniques are ;
1. Q-Banding or Fluorescent Banding.
(a) with quinacrine mustard (QM) or quinacrine dihydrochloride (Q)
(b) with Hoechst 33 258
(c) with chromomycin and DAPI.
(d) D-bands with antibiotics
(e) Ethidium bromide as counter stain for Quinacrine with other chemical
2. G- Banding or Giemsa banding /GTL Banding
(a) with Giemsa
(b) with feulgen.
3. C- Banding
(a) CT- Banding
(b) Cd- Banding.
4. R- Banding or reverse banding
(a) Giemsa Reverse banding
(b) R- Banding by Fluorescence using a cridine orange (AO)
5. T-Banding
6. O-Banding or orcein banding
7. N-Banding /NOR staining (Silver Nuclear organizing region staining)
8. Hy- Banding.
9. Restriction enzyme banding.
The bandings containing heterochromatin or histone-DNA-complexes, respectively, can easily be stained with conventional chromosome dyes and are thus simple to identify.
CONVENTIONAL GIEMSA STAIN-1
PRINCIPLE
Conventional staining techniques are used to uniformly stain chromosomes and leave the centromers constricted, thus enabling measurement of chromosome length, centromeric position, and arm ratio.
BACKGROUND
Prior to 1960, when Moorehead and Nowell described the used of Giemsa in their chromosome preparations, conventional cytologic stains such as acetoorcein, acetocarmine, gentian violet, hematoxylin, Leishman’s, Wright’s, and Feulgen stains were used to stain chromosomes. The Romanovsky dyes (which include Giemsa, Leishman’s, and Wright’s stain) are now recommended for conventional staining, because the slides can be easily distained and banded by most banding procedures. Orcein-stained chromosomes cannot be distained and banded; therefore, orcein is generally not used in routine chromosome staining. Giemsa stain is now the most popular stain for chromosome analysis (Gustashaw, 1991).
SOLUTIONS,
1. Giemsa stain,
2. PH 6.8 phosphate buffer
3. Working stain: 4ml Giemsa; 96ml pH 6.8 buffer.
PROCEDURE
1. Place slides in a Coplin jar or staining dish.
2. Prepare the working stain and pour it over the slides.
3. Stain for 7 minutes.
4. Rinse slides in two changes of distilled water.
5. Air dry slides; mount them with a cover slip if desired. (If sequential banding procedures are to follow, cover slipping is not recommended.)
CONVENTIONAL GIEMSA STAIN-2
SOLUTIONS,
1. 5N HCI,
2. Distilled water,
3. PH 6.8 phosphate buffer
4. Giemsa stain: 2 ml stock Giemsa; 4 ml pH 6.8 buffer; 92 ml distilled water
PROCEDURE
1. Place slides in a Coplin jar containing 5N HCI for 10 minutes at room temperature.
2. Rinse with tap water for 10 minutes.
3. Stain for in Giemsa for 10 minutes.
4. Rinse, dry, and coverslip, if desired.
LEISHMAN’S STAIN
SOLUTIONS,
1. Leishman’s stain
2. pH 6.8 phosphate buffer
3. Working stain: Leishman’s stain diluted 1:4 with buffer.
4. Xylene.
PROCEDURE
1. Stain slides 3to 5 minutes in working stain.
2. Rinse well in buffer. If satin is too intense, wash longer in buffer. If it is too weak, restain the slides.
3. Blot dry with bibulous paper. Air dry completely.
4. Rinse in xylene.
5. Mount in neutral mounting medium
FLUORESCENT BANDING OR Q-BANDING
In a series of papers published during 1968-70, T. Caspersson and his co-workers from Stockholm demonstrated that quinacrine mustard staining produces characteristic bright and dark bands on chromosome. Q-bandings result after treatment of the chromosomes with the fluorochrome quinacrine (= atebrin) or quinacrine mustard. They can be recognized by a yellow fluorescence of differing intensity. Most part of the stained DNA is heterochromatin. Quinacrin (atebrin) binds both regions rich in AT and in GC, but only the AT-quinacrin-complex fluoresces. Since regions rich in AT are more common in heterochromatin than in euchromatin, these regions are labeled preferentially. The different intensities of the single bands mirror the different contents of AT.
Comings et al. (1975) have suggested that quinacrine mustard binds to chromatin by intercalation of the three planar rings with the large group at position nine lying in the small grove of DNA. Most pale staining regions are caused by a decreased binding of Q, predominantly due to non-histone proteins. DNA-base composition influences the fluorescence by correlation with G-C bonding at lower Q: DNA ratios and by conversion of dyes bound near G-C bases into energy sinks at higher ratios. Chromosomal proteins possibly have a much less pronounced effect (Latt, Brodie and Monroe, 1974).
Other fluorochromes like DAPI or Hoechst 33258 lead also to characteristic, reproducible patterns. Each of them produces its specific pattern. In other words: the properties of the bonds and the specificity of the fluorochromes are not exclusively based on their affinity to regions rich in AT. Rather, the distribution of AT and the association of AT with other molecules like histones, for example, has an impact on the binding properties of the fluorochromes.
CHEMICAL ASPECTS,
The structure of quinacrine mustard dihydrochloride (QM) and quinacrine dihydrochloride (Q) are shown below in fig-3 & 4.
The chemical principle underlying Q-banding has been well worked out. With quinacrine mustard (QM), the fluorescent amino-acridine nucleus becomes intercalated within the double helix of DNA. The basic nitrogen atoms form ionic bonds with DNA phosphate and the alkylating side group binds covalently with guanine from DNA (Modest and Sengupta, 1973). Quinacrine dihydrochloride or atebrin does not form a covalent link due to the absence of alkylating side group. Thus the primary Q binding with both compounds has been suggested to be through intercalation of the acridine nucleus in the double helix.
The binding of quinacrine mustard, as opposed to that of quinacrine, is relatively irreversible, in that ethanol or dilute acid under mild conditions will extract quinacrine but not quinacrine mustard from the acridine-DNA complex.
(a) with QM & Q
Principle:
Chromosomes are treated with quinicrine mustard solution, a fluorescent stain, to identify specific chromosomes and structural rearrangements. It is especially useful for distinguishing the Y chromosome (also Y bodies in interphase nuclei) and various polymorphism involving satellites and centromers of specific chromosomes.
The method of Q-banding has undergone a lot of modifications from the time when it was first applied by Caspersson, Lomakk and Zech in 1971 and then Zech alone in 1973 and then Schester in 1975. The modified Q-banding technique is mentioned here.
Equipments:
Mettler analytical balance;
3 one-liter bottles;
8 Coplin jars;
10ml serological pipette;
50ml beaker;
Clean coverslip;
Reagents:
Quinicrine mustard dihydrochloride, 25mg. Store desiccated in light-free container at 20-degree C.
Dibasic sodium phosphate, reagent grade.
Citric acid, anhydrous reagent grade.
Sucrose, reagent grade.
Quinicrine mustard (QM) solution: dissolve 2mg of QM in 10ml distilled water and dilute to 40ml with McIlvanes buffer with a final concentration of 50mcg/ml. Store in Coplin jar (light-free) 1-2 weeks in refrigerator.
McIlvanes buffer (pH 7.0)
(a) Stock solution A: dissolve 28.38g of dibasic sodium phosphate in 900ml of distilled water; then add water up to one liter. Store in tightly capped bottle in refrigerator.
(b) Stock solution B: dissolve 21.01g of citric acid in 900ml of distilled water; then add water up to one liter. Store the same as solution A.
(c) McIlvanes buffer working solution: to make 200ml of solution at pH 7.0, combine 164.7ml of A with 35.3ml of B.Mix well. Store in refrigerator in tightly capped bottle.
Sucrose syrup: dissolve with heat 6g of sucrose in 10ml McIlvanes buffer (pH 7.0), then store in tightly capped bottle in refrigerator.
Procedure:
1. Place dry, slide in 0.2 N HCI for one hour. After ½ hour turn on pre-set waterbath and start to filter Ba(OH) 2 through no.1 Whatman filter paper into Coplin jar.
2. Rinse slide (treated in 0.2 N HCI) in Coplin jar filled with distrilled water.
3. Place rinsed slide in freshly filtered Ba(OH) 2 solution for two minutes.
4. Rinse with distilled water in squirt bottle (some force is required to remove Ba(OH) 2 crystals.
5. Place rinsed slide in Coplin jar (in water bath) filled with 2X SSC (Sodium Saline Citrate) at approximately 62.5 degrees C for one hour.
6. Remove slide slowly and rinse gently in Coplin jar filled with freshly distilled water.
7. After drying, the slide should be stained as follows:
-For peripheral blood specimen, stain 90 seeconds with 1:5 Wright’s stain (For specific information about stain preparation, see G-banding procedure).
This is what we spoke about Q-banding with Q or QM. Now the method of Q-banding with Hoechst 33258 are mentioned here.
(b) With Hoechst 33258
Hoechst 33258 (2-[2-(4-hydroxyphenyl)-6-benzimidazolyl]-6-(1-methyl-4-piperazyl)-benzimidazole), a bibenzimidazole derivative, shows enhanced fluorescence with both AT and GC-rich DNA. However enhancement of AT-rich DNA is greater (Weisblum and Haenssler, 1974) and it can be used as a probe for identifying all types of AT-rich regions in chromosomes, including those which are not demonstrable with quinacrine (Rowley and Bodmer, 1971; Hilwig and Gropp, 1972; Seth and Gropp, 1973). Its interaction with DNA and chromatin is characterized by changes in absorption and circular dichroism measurements (Latt and Wohlleb, 1975). Comings (1975) suggest that H33258 binds by an attachment to the outside of the double DNA helix interacting with base pairs. This binding allows greater sensitivity to the base composition than found with intercalating agents. It has been employed in the study of spontaneous and induced chromosome aberrations (Raposa and Natarajan, 1975) and in mouse-human heterokaryon analysis as an alternative to auto radiography (Moser, Dorman and Ruddle, 1975).
Hoechst fluorescence by heating in plants:(Filion et at., 1976).
1. Fix root tips, after pre-treatment in colchicines (4h), in acetic-ethanol (1:3),
2. Threat 1 day old slides in barium hydroxide/saline mixture (Filion, 1974)
3. Stain in H33258 (40 µg/ml) in McIlvaine’s buffer, pH 4.1, for 10 s.
4. Rinse and mount slides in same buffer and seal.
5. Heat the sealed slides for about 6 s on a hot plate (120ºC) and cool them rapidly by placing them between two slabs of dry ice.
The chromosome banding increases markedly in fluorescent intensity as compared with control. This method however is not applicable to all plants. A combination with Giemsa banding has been employed in rye (Vosa, 1974).
In Drosophila chromosomes (Holmquist, 1975).
1. Dissect out ganglia in Ringer’s,
2. Incubate 45 min at 24 ºC in Schneider’s cell culture medium with 0.05-µg/ml colcemid.
3. Keep in 1 percent trisodium citrate for 10 min, 0.5 per cent trisodium citrate for 5 min.
4. Fix in three changes of acetic-methanol (1:3) for 30 min.
5. Warm to 50 ºC in a drop of 60 per cent acetic acid.
6. After 30 s, cells dissociate from the mass of tissue and adhere to the glass at the edge of droplets.
7. Add a drop of (1:3) fixative to warm slide.
8. Rinse slides for 10 min in PBS (0.15 M NaCl, 0.03 M KCI, 0.01 M Na phosphate pH 7.0),
9. Stain 10 min with a 1/100 dilution of stock 33258 Hoechst in PBS,
10. Rinse 10 min in PBS
11. Rinse 4 min in water,
12. Drain and store for less than 12 h.
13. Mount in McIlvaine’s buffer pH 4.0 and observe in fluorescence microscope.
(C) with Chromomycin and DAPI.
Two DNA binding guanine specific antibiotics, chromomycin A3 (CMA) and closely related mithramycin (MM) are used as fluorescent dyes. Metaphase chromosomes in roots were sequentially stained with CMA or MM and the DNA-binding AT-specific fluorochrome 4́-6 diamidino-2 phenylindole (DAPI). Non-fluorescent counterstain like methyl green and actinomylin D (AMD) may be used with CMA and DAPI respt. In general, C-bands, which are bright with CMA and MM, are pale with DAPI and vice versa. CMA-banding appears to resemble R-bands.
Preparation of slides for staining:
1. Treat root tips with colchicines (0.05%, 3-6 h)
2. Fix in acetic. Ethanol (1:3, overnight)
3. Squash in 45 per cent acetic acid.
4. Air dry after removing coverslip by dry ice.
For human or mammalian chromosome air dried preparation is used.
CMA STAINING:
1. Pre-incubate slides in McIlvaine citric acid- Na2 HPO4 buffer (pH 6.9-70), containing 10 mm MgCl2 for 10-15 min.
2. Stain slides in buffer containing 10 mm MgCl2, and 0.12 mg/ml CMA (‘reinst’, Serva, Heidelberg) or 0.11 mg/ml MM (‘rein’, Serva) for 5-10min.
3. Wash and mount in McIlvaine’s buffer (pH 6.9-7.0) and seal with rubber solution.
For counterstaining,
1. Pre-incubate slides in McIlvaine’s buffer (pH 4.9) for 10-25.
2. Stain for 5-15 min in a buffered solution (pH 4.9) of 0.1 per cent methyl green GA (Chroma, Stuttgart), from which methyl violet has been removed with chloroform extractions.
3. Rinse in buffer (pH 4.9) and in neutral buffer containing 10 mm MgCl2,
4. Stain in CMA as described before.
AMD + DAPI DOUBLE STAINING;
1. Dissolve AMD in small amount of methanol and make up to 0.25 mg/ml with McIlvaine’s buffer (pH 6.9-7.0).
2. Pre-incubate human chromosome slides in buffer for 5 min.
3. Stain with AMD for 15-20 min,
4. Rinse with Mcilvaine buffer (pH 6.9-7.0).
5. Stain in DAPI at concentrations between 0.1 to 0.4 µg/ml for 5-10 min.
CMA and MM fluorescence fade rapidly and can be slightly stabilized by ageing the preparation and the solution.
DAPI fluorescence is observed with excitor filters UG 1, BG 3 and TK 300 (dichroic mirror of Leitz Ploem epi-illuminator) and barrier filters K-400 of the epi-illuminator and K-470, K-490 or K-510.
DAPI / DISTAMYCIN A STAINING
Background:
The DAPI/ distamycin A fluorescent staining technique was first described by Schweizer, Ambros, and Andrle as a method for labeling a specific subset of C bands. (Gustashaw, 1991).
Principle:
The DAPI/Distamycin A staining technique is useful in identifying pericentromeric break points in chromosomal rearrangements and in identifying chromosomes that are too small for standard banding techniques. Also, DAPI/DA is the method of choice for Yqh chromosome material in suspected Y autosome translocations.
Stains
Distamycin A:
1. Dissolve 2 mg distamycin A-HCI (Sigma) in 10ml of McIlvaine’s buffer, pH 7.0 Add a magnetic stirrer and place the foil-convered tube in a beaker of crushed ice. Stir for 15 to 30 minutes. Dispense in 1 ml aliquots and store at 20 degrees C for up to six months.
2. DAPI Stock solution: Disslove 2 mg DAPI-2 HCI (Sigma) in 10 ml of distilled water. Dispense in 1 ml aliquots, and store at-20 degrees C for up to six months.
3. Working solution: Add 0.1 ml of stock solution to 100ml of McIlvaine’s buffer, pH 7.0. Store at 4 degrees C for up to six months.
4. Buffer: McIlvaine’s buffer, pH 7.0.
Solution A : 0.1 mol/L citric acid (19.2g:q.s. to 1 liter with distilled water)
Solution B:0.2 mol/L Na2HPO4 (28.4g:q.s. to to 1 liter with distilled water)
The following formula can be used to prepare this buffer at various pHs:
x ml A+(100-x) ml B=100ml total volume.
For pH 4.1, x = 60
For pH 5.5, x = 43.1
For pH 7.0, x = 18.2
Procedure:
1. Flood a slide with distamycin solution. Coverslip and incubate the slide, in the dark, at room temperature for 5 to 15 minutes.
2. Remove the coverslip and rinse briefly with pH 7.0 buffer.
3. Flood with DAPI working solution. Coverslip and incubate the slide, in the dark, at room temperature for 5 to 15 minutes.
4. Remove the coverslip and rinse the slide briefly with pH 7.0 buffer.
5. Observe with fluorescence (excitation:340-380 nm; suppression: 430 nm).
6. Photograph as for other fluorescent techniques.
Precautions:
Use care when handling these substances. Wear gloves. Distamycin A tends to lack stability in aqueous solution unless it is frozen. DAPI stock solution can be frozen or refrigerated for several weeks without detectable deterioration. The stained preparations tend to fade rapidly, so photography should be immediate. Storing coverslipped slides at 4 degrees C for a day or so may stabilize the fluorescence.
d) D- bands with Antibiotics
Of the anthracyclin group of antibiotics, daunomycin (Cerubidine-HCI) and adriamycin (Doxorubicin-HCI) give well-defined and reproducible orange-red fluorescent banding patterns on human chromosomes (Lin and de Sande, 1975). The concentrations are 0.5 mg for daunomycin and 0.2 mg for adriamycin per ml in 0.1 m sodium phosphate buffer (ph 4.3) and the staining period 15 min. The schedule and pattern are similar to Q-bands. The G-C specific DNA binding antibiotic-olivomycin produces reverse fluorescence banding patterns in human, bovine and mouse chromosomes (de Sande, Lin and Jorgenson, 1977).
e) Ethidium bromide as counterstain for quinacrine
(Hollander, Litton and Liang,1976).
1. In case of plants, the cellular smear is fixed in 95 percent ethanol. While in case of animals materials, air dried preparation are used.
2. Keep in 95 percent and 70 per cent ethanol for 3 min each; distilled water, 3min; citric acid phosphate buffer (0.01 M, pH 5.6), 3 min; staining solution (50µg/ml QM in citric-phosphate buffer 0.01M, each in pH 5.6 buffer; 0.5 percent zinc sulphate), 10min; two changes of 6min each in pH 5.6 buffer; 5 percent zinc sulphate solution , 5min ;two changes in distilled water of 4 min each; ethidium bromide solution (2µg/ml EB in 7.0 pH phosphate buffer 0.01M),5 min 7.0 pH phosphate buffer (0.01M) two changes of 2 min each, 7.6 pH phosphate buffer (0.01M), two changes of 4 min each mount in same buffer and seal.
With this counter stain, cytoplasm fluoresces pale green, nuclei pale orange and interphase fluorescent bodies appear as bright yellow spots as also do the brightly fluorescing bands in metaphase chromosomes.
f) Other chemicals:
Other chemicals have been observed to give quinacrine-like fluorescence as well. These include alcoholic extracts of the alkaloids from fresh roots of eight genera of Papaveraceaee and Fumariaceae (Vosa et al.,1972) as also from Chelidonicum majus, Macleaya cordata and Glacium flavum. Of limited use are Sarcolysinoacridine (Iordanskiy et al.,1971), Berberine sulphate (Moutschen Degraeve and Moutschen-Dahmen, 1973) and 2,7-di-t-butyl proflavine DBP (Disteche and Bontemps, 1974).
Frog chromosome do not bind with quinacrine/mitramycin/chromomycin A3 (Schmid, 1980). Also as opposed to avian and mammalian DNA, amphibian (Xenopus and Pleurodeles) DNA lacks the base composition isochors , long DNA stretches of homogeneous base composition, and have little DNA in the ‘GC-rich’ region of the density gradient where mammalian R-band DNA is found (Macaya et al., 1976; Bernardi et al.,1985). Thus the quinacrine bandability of mammalian chromosomes evolved since mammals diverged from amphibians, it is directional, it is due to an increased dG and dC content of R-band DNA, and it may be a directional response to homeothermia (Bernardi and Bernardi,1986; Bernardy et al., 1985 .).The homeothermia cause implies Q-bandability evolved twice independently in birds and mammals, and would require showing that reptiles do not have the isochores required for Q-bandability of their chromosomes.
GIEMSA BANDING OR
GTL BANDING
This technique was developed by Tau-Chiuh Hsue and Frances E.Arrighi. It is observed that when the chromosomes are incubated in saliva are stained with Giemsa stain or treated with urea or detergents, G-bands appear in the areas which are S-rich proteins. Q & G- bands are identical. Most laboratories prefer G-banding for routine purposes since no fluorescence microscope is needed & the slides preserve the dye on storage.
Giemsa is a complex mixture of thiazine dyes and eosin .Of them, methylene blue and Azure A,B and C alone give good banding. Thionin, with no methyl groups, gives poor banding while eosin has no effects. Thiazine dyes are strongly metachromatic and their adsorption spectra and extinction coefficients change with increase in dye concentration or on binding to positively charged chromotropes (Comings,1975). Dye concentration, according to Walther, Stengel-Rutkowski and Murken (1974) is more important than trypsin digestion or denautration.
According to Matsui Sasaki (1975) during G-banding, macromolecules like DNA and proteins are lost, leading to an uneven distribution of chromatin. Non- histone proteins of relatively larger molecular sizes are removed by banding methods. The residual proteins in the G-bands are relatively small molecules containing a large number of S-S bonds and possibly contain more stablised DNA. Thus the G-positive bands represent relatively thermostable chromatins consisting of smaller nonhistone protein molecules. At the same time polylysine, polyarginine and histone H1,H2A ,H2B and H3 inhibit G-staining and chromosome banding by binding to DNA and preventing side stacking of the positively charged thaizine dyes to the negatively charged phosphate groups on DNA.
G-banding is well suited for animal cells but are not of very usefull with plants. Plant chromosome treated with this technique are uniformly stained.
(a). With Giemsa.
The method of G-banding which was successfully used in the Cytogenetics Laboratory of the Laboratory of Pathology in Seattle, Washington, USA is given below.
Chromosomes are G-banded to facilitate the identification of structural abnormalities. Slides are dehydrated, treated with the enzyme trypsin, and then stained.
Glass coplin jars with lids; 5ml and10ml serological pipettes and bulbs; Staining rack; Timer; Plastic squirt bottle; Dehydrating oven set at 95 degrees C; Slide racks; Slide warming tray set at 60 degrees C;
0.25% trpsin,in HBSS without calcium and magnesium. Store at – 5 to -20 degrees C. Thaw at room remperature -- do not heat above room temperature to avoid denaturing the enzyme.
Dry salt phosphate buffer, pH 6.8
Leishman’s stain powder.
Absolute methanol. Toxic -- avoid contact with skin and mucous membranes. Flamable --store in approved fire – proof cabinet. DDispose down fume hood sink with copius amounts of water.
Normal saline, 0.9% sodium chloride.
pHydrion buffer capsules, pH 7.0
0.025% trypsin: mix 5ml 0.25% trypsin with 45ml normal saline. Prepare fresh the day of staining. Keep at room temperature. Do not use if color changes.
Fisher phosphate buffer: dissolve 1 buffer capsule (pH 6.8) in 1 liter of distilled water.
Check pH with pH meter and record on the label.
Stain : swirl 500ml absolute methanol in flask. Add 1.0g powdered stain to swirling methanol. Continue to swirl 2-3 minutes at moderate rate. Let sit for 15 minutes. Filter through Whatman #1 filter paper into brown bottle that contains no water (swirl bottle with methanol before use). Store away from heat and light. Shake well and filter before use.
pHydiron stock buffer: dissolve 1 pHydrion buffer capsule (pH 7.0) in 100ml distilled water. Check pH with pH meter and record on label. Store at 2 to 8 degrees C.
pHydrion woring solution : mix 5ml pHydrion stock buffer with 95 ml of distilled water.
Store at room temperature.
References
Seabright, M. Rapid banding techniques for human chromosomes. Lance 2: 971-972, 1971
Franke, U., Oliver, N. Quantitative analysis of high- resolution trypsin-Giemsa bands on human prometaphase chromosomes. Hum. Genet. 45:137-165, 1978.
(b). With feulgen stain.
Feulgen staining has been employed in lieu of Giemsa in producing G-bands as described above.
Prolonged hydrolysis and feulgen staining in species of Chilocorus produce banding patterns opposite to Q-banding (Ennis,1975).
Procedure for plants. (Merritt, 1974).
1. Store pre treated fixed root tips in 70 percent ethanol at 4ºC for at least four days.
2. Hydrolyse in 10 percent HCl at 60ºC ,
3. Wash thoroughly,
4. Stain in leucobasic fuchsin solution,
5. Wash and stain in aceto-carmine for 5 min.
6. Squash in a drop of aceto-carmine .
7. Heat over a steambath for 1 to 2 min.
This process has limited application.
This is one of first chromosome banding technique discovered by Pardue and Gall as a by product of the in situ RNA/DNA hybridization (Pardue & Gall,1970) the name is derived from centromeric or constitutive heterochromatin.
The preparation undergo alkaline denaturation prior to staining leading to an almost complete depurination of the DNA after washing the probe the remaining DNA is renatured again and stained with Giemsa solution consisting of methylene azure, methylene violet, methylene blue, and eosin. Heterochromatin binds a lot of the dye, while the rest of the chromosomes absorb only little of it(Hsu,1973;Arrighi, 1974). The C-banding proved to be especially well-suited for the characterization of plant chromosome but the method was initially devised for mammalian chromosomes.
Staining of constitutive (or centromeric) hetrochromatin ( not facultative hetrochromatin like inactive X chromosome) using Giemsa was first observed in mouse chromosomes.
The application of banding techniques to plant materials was initially limited because of technical difficulties. However, species specific Giemsa C-banding techniques were later developed for several crops including Vicia faba, Secale cereale (rye) and other species of Secale, Hordeum vulgare (barley)and other Hordeum species, Avena species, Triticum aestivum, X Triticosecale, etc. The original technique of Giemsa C-banding involved the following steps: (i) prepare the slide for chromosomes through conventional squash technique; (ii) treat the slide with 0.2N HCI for 10-30 min; (iii) wash the slide in deionized water; (iv) treat the slide with NaOH (2X SSC with pH adjusted to 12) for 2 min ; (v) rinse in ethanol several times; (vi) incubate in 6 X SSC over night at 60ºC (SSC= Sodium saline citrate); (vii) stain in Giemsa solution; (viii) wash and mount in DPX.
The recent method of C-banding which was successfully use din the Cytogenetics Laboratory of the Laboratory of Pathology in Seattle, Washington USA is as follows.
Principle:
To specifically stain the centreomeric regions and other regions containing constitutive heterochromatin, i.e., the secondary contrictions of human chromosomes 1,9,16, and the distal segment of the Y chromosome long arm.
Equipment:
Circulating waterbath set at 65 degrees C;
Four glass Coplini jars with lids;
9.0cm circles of Whatman # 1 filter paper;
Squirt bottle filled with distilled water;
Slide warmer;
Timer;
Reagents:
Xylene substitute: Toxic-avoid contact with skin and inhalation. Store in approved fire-proof cabinet. Discard in organic waste container.
Absolute methanal: Toxic-avoid contact with skin and inhalation. Store in approved fire- proof cabinet.
Glacial acetic acid: Toxic- avoids contact with skin and inhalation .Store in approved fireproof cabinet.
Concentrated hydrochloric acid: Toxic avoid contact with skin and inhalation. Store in approved fireproof cabinet.
Sodium chloride crystal
Sodium citrate crystal.
Barium hydroxide crystal.
3:1 Fixative: three parts absolute methanol with one part glacial acetic acid. Discard extra fixative down fum hood sink with copius amounts of water. Prepare fresh.
0.2N HCI: 4.15 ml concentrated hydrochloric acid to 200ml of distilled water, mix and add water to 250ml. Stable 1 year.
2 X SSC: dissolve 17.53g sodium chloride crystal and 8.82g sodium citrate crystal in one liter of distilled water. May be refrigerated in thightly-capped bottle indefinitely.
2.5% Ba(OH)2:dissolve 2.5g of barium hydroxide crystal in 100ml distilled water. This is a supersaturated solution. Store in thightly capped bottle at room temperature. Stable indefinitely.
Procedure:
1. Place dry, slide in 0.2 N HCl for one hour. After ½ hour turn on pre-set waterbath and start to filter Ba(OH)2 through # 1 Whatman filter paper into Coplin jar.
2. Rinse slide (treated in 0.2N HCl) in coplin jar filled with distilled water.
3. Place rinsed slide in freshly filtered Ba(OH)2 solution for two minutes.
4. Rinse with distilled water in squirt bottle (some force is required to remove Ba(OH)2 crystal).
5. Place rinsed slide in coplin jar (in water bath ) filled with 2X SSC at approximately 62.5 degrees C for one hour.
6. Remove slide slowly and rinse gently in coplin jar filled with freshly distilled water.
7. After drying the slide should be stained as follows:
–for peripheral blood specimen, stain 90seconds with 1:5 Wrights stain (For specific information about stain preparation, see G-banding procedure).
References:
Summer, A.A simple method for demonstrating centromeric heterochromatin. Exp. Cell res.75: 304-306,1972.
(a) CT-BANDING
CT- banding is a further amplification of C-banding techniques in human material, treat air dried preparations with Ba(OH)2 solution at 60ºC, incubate in 2X SSC at 60ºC and stain in cationic dye ‘stains all’ (Scheres,1976; Scheres, Hustinx and Rutten,1974).The bands are of C-and R-types , located mainly at telomeric regions.
(b)Cd – BANDING
Cd-banding reveals two identical dots (centromeric dots Cd) at the centromeric region, one for each chromatid (Eiberg,1974). Store mammalian air-dried preparation for one week at room temperature. Include in Earle’s BSS medium (PH 8.5 to 9.0) at 85ºC for 45 min stain in 4 percent Giemsa in 1/300 M phosphate buffer (pH 6.5).
REVERSE BANDING
R-bands show a pattern that is reverse of G-bands i.e. light banded region of G-banded chromosomes became darkly stained & vice versa. R-bands are the results of a technique that stains regions rich in GC that are typical for euchromoatin. R-bands correspond too the regions on chromosome having proteins lacking sulphur.
R-banding was initially developed for human chromosome (Ditrillaux and Lejeune 1971; Carpenter, Ditrillaux and Lejeune, 1972; Ditrillaux, 1973), in which the pattern is opposite to Q and G.
The R-bands are obtained when chromosomes are incubated in a buffer at high temp & stained with Giemsa stain.
(a) Giemsa reverse banding (RHG)
Principle:
R-banding methods are useful for analyzing deletions or translocation that involve the telomeres of chromosomes.
Background: Reverse banding using heat and Giemsa (RHG) was first described by Dutrillaux and Lejeune . This technique involves the incubation of slides in hot phosphate bufer with subsequent Giemsa staining . The resultant chromosome pattern shows darkly stained R bands and pale G bands. R bands are GC-rich and the AT-rich regions are selectively, or more readily, denatured by heat, but the GC-rich regions remain intact. This is consistent with the fact that GC-specific fluorochromes also produce a reverse chromosome banding pattern. (In many laboratories, RHG methods have been abandoned in favor of a fluorescent R-banding technique (Gustashaw, 1991)
Solutions:
§ Buffer:
10.0 ml Earle’s balanced salt solutions (EBSS) 10,
0.1 ml 7.5% sodium bicarbonate
89.9 ml distilled water
Place buffer in water bath, and heat to 88 degrees to 89 degrees C.
§ Tap water
§ 2% Giemsa in distilled water
Procedure:
1. Incubate slides in hot EBSS for 10 to 15 minutes. (Fresh slides require 1 to 2 hours. One-day-old slides require 25 minutes. One week old slides require 7 minutes.In general, older slides require less time.)
2. Cool quickly in tap water do not dry .
3. Stain in 2% Giemsa for 10 to 20 minutes.
4. Rinse in Xylene.
5. Rinse in tap water. Air dry.
References
Gustashaw, KM.Chromosomes Stains, in the ACT Cytogenetics Laboratory Manual, Second Edition, edited by M.J.Barch. The Association of Cytogenetic Technologists, Raven Press, Ltd., New York, 1991.
R BANDING BY FLUORESCENCE USING ACRIDINE ORANGE (AO)
Principle
R-banding methods are useful for analyzing deletions or translocations that involve the telomers of chromosomes.
Back ground
Acridine orange was originally used to stain untreated chromosomes, both human and mouse. Bobrow et al. and Baserga and Castoldi independently reported the use of acridine orange to obtain a reverse banding pattern of chromosomes. Acridine orange (AO) is a base composition-independent flurochrome that binds to DNA by intercalation and which gives relatively uniform fluorescence along the length of the chromosome arms. The dye binds very little to non-nucleic acid cell components, but it fluoresces orange- red when bound to single stranded nucleic acids and yellow- green when bound to double- stranded nucleic acids. Following hot phosphate buffer treatment, R bands are yellow- green and G/Q bands are orange red.The major factor that contributes to R banding is the relative GC-richness of the R bands. In many laboratories, RHG methods have been abandoned in favor of a fluorescent R banding technique (Gustashaw, 1991).
Solutions
§ Phosphate buffer:
32 ml of 0.07N Na2HPO4. 12 H2O
68 ml of 0.07mol/L KH2PO4
Adjust pH to 6.5 by adding 0.07N Na2HPO4. 12 H2O to the solution.
Acridine orange: 0.01%, prepared in the phosphate buffer.
Procedure
1. Prewarm the buffer to 85 degrees C.
2. Incubate one slide for 10 to 30 minutes in hot phosphate buffer.
3. Stain with 0.01% acridine orange for 4 to 6 minutes.
4. Rinse in phosphate buffer (pH 6.5) for 1.5 to 3 minutes.
5. Mount with the same buffer, with out sealing the coverslip.
6. Examine by fluorescence microscopy using the appropriate filter combination (e.g., excitation: 450-490 nm; suppression: 515 nm).
References
Gustashaw, KM.Chromosomes Stains, in the ACT Cytogenetics Laboratory Manual, Second Edition, edited by M.J.Barch. The Association of Cytogenetic Technologists, Raven Press, Ltd., New York, 1991.
Principle
T-banding is used to stain the telomeric regions of chromosomes for cytogenetic analysis.
Background
Telomeric (or terminal) banding was first reported by Ditrillaux, who used two types of controlled thermal denaturation followed by staining with either Giemsa or acridine orange. 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).
T banding by Thermal Denaturation: Method 1
1. Bring 94 ml of distilled water and 3 ml of phosphate buffer (pH 6.7) to 87 degrees C in a coplin jar.
2.Add 3 ml of Giemsa stain.
3.Add slides to jar ; stain for 5 to 30 minutes
4.Rinse in distilled water, air dry, and examine.
For Fluorescent Observation
5.Destain, rehydrate through a series of alchohols, rinse in distilled water.
6.Stain in acridine orange (5mg/100ml) for 20 minutes
7.Rinse in phosphate buffer, mount, and examine with a fluorescence microscope (excitation : 450-490 nm ; suppression :515nm).
Notes
Standard culture methods are appropriate. Slides should be aged for a few days prior to staining. With Giemsa (steps 1-4), the chromosomes are pale, unbanded, and difficult to see. With acridine orange staining for various lengths of time, the chromosomes appear as follows: 45 minutes: green at telomeres, otherwise orange; 15 to 20 minutes: green at telomeres, orange areas are less intense; 30minutes or more: orange color is gone, intercalating R bands appear.
T banding by Thermal Denaturation: Method 2
1. Bring a coplin jar containing Earle’s BSS, PBS, or phosphate buffer to 87 degrees C. The pH must be adjusted to 5.1.
2. Stain with Giemsa or acridine orange as in Method 1, step 2 to 7.
References
Ditrillaux B. Nouveau systeme de marquage chormosomiques: Les bands T. Chromosoma 41:395-402,1973.
Gustashaw, K.M. Chromosome Stains. In The ACT Cytogenetics Laboratory Manual. Second Edition, edited by M.J.Barch. The Association of Cytogenetic Technologists Raven Press Ltd, New York, 1991.
It was developed primarily for use in plant chromosomes (Sharma, 1975,1977), since in plants G, C and Q bandings have relatively limited application. The inherent disadvantages are the securing of air drying of solid tissues and the comparatively lower response to Giemsa reaction.
The technique principally involves an elimination of denaturatuion and consist of pre- treatment of the tissue, fixation in acetic acid-ethanol treatment in a strong concentration of 1X SSC at room temperature (27-28ºC), washing, staining in acid-orcein and mounting in 45 percent acetic acid. Orcein-positive bands appear in different segments of chromosomes, including the centromeric and intercalary ones. The mechanism of reaction possibly involves the DNA- protein linkage, since orcein is an amphoteric dye, capable of staining both DNA and protein. The gradual removal of non-histone protein through SSC treatment is principally responsible for O-banding at the sites of stronger DNA- protein linkage. As the removal of protein by SSC application is gradual, mild treatment result in intercalary bands comparable to G-banding whereas strong prolonged treatment ultimately show only C- bands where the linkage is strong due to highly homogeneous repeats. The method therefore allows localisation of major and minor repeated sequences in chromosomes.
Procedure:
1. Pre-treat the tissue in 0.2 percent colchicine for 2 to 3h at 12 to 14ºC (Other pre treatment chemicals may also be used.)
2.Wash in water for 5 min.
3. Fix in acetic acid-ethanol (1:2) for 2-12 h
4. Treat in 45 percent acetic acid for 5 min.
5. Wash in water.
6. Treat in a mixture of 1 M sodium chloride and 0.1 M sodium citrate (1:1) at 27-28ºC for 2-3 h.
7. Wash in water.
8. Warm for a few seconds at 90ºC in a mixture of 2 percent aceto-orcein solution and N HCI (9:1).
9. Keep in the mixture at 27-28ºC for 1 h.
10. Squash under a cover slip in the 45 percent acetic acid and seal.
NOR STAINING OR
(Silver Nucleolar Organizing Region Staining)
N banding has been employed principally in the localisation of nucleolar organizing regions. It has been suggested that these bands represent certain structural non-histone proteins specifically linked to nucleolar organizers in different eukaryotic chromosomes.
This technique was first applied by Matsui & Sasaki, 1973 for human chromosomes. The most recent procedure of NOR-staining was successfully used in the Cytogenetics Laboratory of Laboratory of Pathology in Seattle, Washington ,USA which is given below.
Principle:
Chromosomes are treated with sliver nitrate solution, which binds to the Nucleolar Organizing Regions (NOR), i.e., the secondary constrictions (stalks) of acrocentric chromosomes.
Equipments:
Incubator set at 37 degrees C;
30ml capped amber bottles.
18 gauge 1.5"needles.
1cc tuberculin syringes,
02mcg Whatman #2 Filter paper circles;
Dish, Petri, Kimax, 150mm × 20mm
Small plastic lids or rings;
Squirt bottle, filled with distilled water;
Waterbath set at 37ºC
Glass coplin jar.
Regents:
Silver nitrate
Deionized, distilled water
50% silver nitrate solution: dissolve 5g silver-nitrate in 10ml distilled water and pour into clean an dry amber bottle. Label and store at 4 degrees C.
Procedure:
1. Place unstained slide in coplin jar filled with distilled water. Place in 37 degrees.
C water bath for 2 hours. Remove slide and allow to air dry.
2. Prepare moist chamber by putting two 12.5cm circles of Whatman #2 filter in bottom of glass petri dish. Saturate paper with distilled water. Be sure to remove trapped air bubbles in paper. Place plastic lids or rings on wet filter paper to support each slide at both ends. Cover dish.
3. Attach 18 gauge needle to 1cc syringe.
4. Remove silver-nitrate solution form refrigerator and place bottle in beaker to prevent tipping (see “Precaution” below).
6. Put on gloves
7. Draw 1cc of silver-nitrate solution into syringe.
8. Remove needle and replace with acrodisc filter.
9. Attach unused 18 gauge needle to acrodisc filter.
10. Lay 5-7 drops of filtered solution near labeled end of slide. Return unused solution to storage bottle. Refrigerate. Discard syringe, filter and needle in Sharpgard container.
11. Using forceps lower one end of pre-cleaned coverslip into drops of silver nitrate solution on slide and gently lower coverslip to avoid trapping air bubbles.
12. Transfer treated slide to moist chamber and support on lids above wet filter paper. Cover dish.
13. Carefully place moist chamber in 37 degrees C incubator. Check to see that treated slide is horizontal after closing inner glass door of incubator.
14. Incubate for 7 hours at 37 degrees C.
15. Put gloves back on. Carefully remove slides from moist chamber and using squirt bottle of distilled water wash coverslip and silver nitrate solution into sink (with tap water running). Rinse slide in coplin jar of distilled water. Let dry. Discard coverslip left in sink using forceps. Do not dismantle moist chamber.
16. Check treated slide under 10X phase and 10X phase to judge effectiveness of first treatment.
17. If stained NOR’s are unapparent, repeat steps 3-15 for one hour.
18. Repeat steps 14 and 15.
19. Repeat treatment at on hour intervals two or three times if necessary.
20. Counterstaining is not necessary, but may be desired. Counterstaining with Quinacrine is ideal; however, a plae counterstain with Wright’s stain (see “G-banding” procedure) may work satisfactorily. If Wright’s stain is too dark , it is difficult to distinguish between satellite and silver staining on NOR’s.
Notes and precautions
1. 50% silver-nitrate solution should be handled with care. It is a clear solution initially, thus difficult to detect. If allowed to get on skin, it can cause a chemical burn which turns black. This cannot be washed off. Discoloration disappears only when a new layer of skin is formed. Clothing is also vulnerable to silver nitrate staining.
2. Slides that have been previously G banded are not suitable for NOR staining.
References
Bloom, SE; Goodpasture , C An improved technique for selective silver staining of Nucleolar Organizer Region in human chromosomes. Human Genetics 34:1990296,1976.
Lay, YF, Pfeiffer, RA; Arrighi, F.E; Hsa, TC combination of silver and fluorescent staining for metaphase chromosomes. Am J Hum Genet 30:76-79,1978.
This method developed especially for plant cells. Hy-banding was initially applied to somatic chromosomes of some members of the Liliflorae. Root tips, fixed in acetic ethanol (1:3) , on treatment with 0.1 or 0.2N HCL between 60 and 80ºC and staining with aceto-carmine, gave banded chromosomes (Greilhuber, 1973,1974,1975). The pattern of Hy-bands is different from that of C-bands.Hetrochromatic regions staining differentially, mentioned as HY+ and HY- bands but do not always coincide with G-bands it seems that the binding of protein to DNA & its more or less complete extraction has an impact on the binding ability of the acetocarmine.
OF METAPHASE CHROMOSOME
Treatment with restriction enzymes is an esffective way to produce banding of fixed metaphase chromosomes. Hae III treatment leads to G banding of all mammalian species tested so far. No enzyme has been reported to produce an R-band pattern in any species (Dorothy A. Miller and Orlando J. Miller, 1988).
Type 2 restriction endonucleases or restriction enzymes are derived from various bacteria where they serve to restrict the introduction of foreign DNA. Each enzyme recognizes a specific palindromic DNA sequence four or more base pairs (bp) in length and cuts DNA at this specific site to yield blunt ends or staggered ends each with a short single- stranded region. DNA that has been methylated to form 5-methycytosine or 6-menthyladenosine within the recognition sequence is not cut, providing the bacterium with a mechanism for distinguishing self (specifically methylated) from foreign (unmethylated) DNA at the sites.
Most restriction enzyme recognition sequences are available for cutting in chromatin (isolated nuclei). The recognition sequences are also available for cutting in metaphase chromosomes that have been fixed and air dried on a slide. Restriction enzyme digestion leads to removal of some DNA fragments, which are below a certain critical size; the remaining DNA can be stained to reveal the relative amount and location of the unextracted DNA. Enzymes that cut DNA into fragments whose average length is less than 1000 kb produce banding patterns on metaphase chromosomes. Studies in a variety of species show that restriction enzyme treatment can lead to G-banding or to specific modified C-band patterns, the latter reflecting extraction of major components of some or all C bands. Some enzymes have no effect on chromosome staining.
Restriction enzyme can be classified into three types based on their effect on human chromosome when digestion is carried to completion : type one produces G-banding plus C-banding on some chromosomes, type two yield C-banding on some chromosomes often with a small amount a residual G-banding, and type three produces no banding.
Type three is of less cytological interest. This enzymes are numerous & fall under two groups.
Example of Type-1 restriction enzyme: -
Hae III produces clear G-banding on numbers 1,9,16 the Y & a few acrocentric chromosomes (Mezzanotle et al.,1983, Miller et al., 1983; M. Bianchi et al.,1985; Babu & Verma, 1987a)
Example of Type-2 restrictor enzymes: -
MboI leaves intact the large C-band on number 9, & smaller C bands on a variety of chromosomes, (Miller et al., 1983; M. Bainchi et al., 1985).
Example of Type-3 restriction enzyme: -
MspI doesnot produce a visible banding pattern throughout the genome (Miller et al.,1983).
Restriction enzyme banding occasionally reveals additional differences in the C-bands region of homologous chromosomes (M. Bianchi et al.,1985)
1. The banding pattern techniques have brought a revolution in the study of karyotype insofar as they permit precise identification of individual chromosome segments, with fat reaching consequences. Not all of them are reliable & certainly none can be applied to all organisms.
2. Chromosome banding technique in its present form, is a synthetic procedure permitting the visualization of molecular sequences at the cellular, microscopic level.
3. Many scientists have worked to find evolutionary relationship between different species by observing banding patterns. In some cases they could relate while in other they could not. Some of their observation are as follows: -
a). Species within a family generally show considerable matching of chromosomes banding patterns. The cat, camel & cow families each have interfamilial similarities in chromosomal banding patterns.
b). On the other hand, some species have banding patterns which differ greatly from those of other species in the same genus. Similar species with different chromosomal banding patterns are found among certain deer (the muntjacs) & bats (family Phyllostomidae).
c). Similarities in chromosomal banding patterns are observed among species, which are different in structure. Interfamilial chromosomal banding similarities are found amount the cats, mongoose, & racoons; among the cow, deer, & giraffe families ; among several families of marsupials ; & among several families of primates, including humans
4. By identifying the isochores of the chromosome by banding technique, the related species can be identified. Isochores consists of a mosaic of very long (>300 kb) DNA segments. (Bernardi et al., 1985; Bernardi & Bernardi , 1986)
5. Many non-modefied attempts of chromosome banding are well suited for animal chromosomes but non for plant chromosomes. The banding pattern of plant chromosomes does never reach the same degree of resolution common with animal chromosomes. In the coming days the techniques should be modified so that they suit well for plant chromosomes too.
6. Different scientific have undertaken comparative study of different banding techniques in chromosomes of different species which were found effective & usefull in identification of translocations of chromosomes etc.
7. By chromosomal banding technique the chromosomal aberration of the aberrated chromosome can be identified which is very useful for the treatment of patient.
1. A.T.Summer (1976) Banding as a level of Chromosome Organization. Current Chromosome Research.( Editor- K.Jones & P.E.Brandhan) Proceeding of the key chromosome conference held at the Jodrell Laboratory. The Royal Botanic Gardens, Kew, England. North Holland Publishing Company. New York.17-22
2. Arun kr. Sharma and Archana Sharma. Chromosome Techniques. Theory and Practical (3rd edition), Butterworths & Co. 408-37
3. C.D. Darlington and L.F.La cour. The handling of Chromosomes (6th edition) Georgia Allen and UnwiN Ltd. London.61-62,131-133.
4. Dorothy A. Miller and Orlando J. Miller (1988b) Restriction Enzyme Banding of Metaphase Chromosomes. Trends in Chromosome Research (Editor-T.Sharma)- Verlag Narosa Publishing House.238-49.
5. Edward.J.Modest and Sisir.K.Sengupta (1973). Chemical Aspects of the Fluorescence Analysis of Chromosomes. Fluorescence techniques in Cell Biology. Editors -A. A.Thaer & M. Sernetz. Springer Verlag New York. 125-33
6. F. Vogel and A.G Motulsky (1979) Human Genetics- Problems and Approaches. Springer-Verlag. New York.
7. Gerald P. Holmquist (1988b) . Contents of G- and R- bands Defy Contemporary Paradigms. Trends in Chromosome Research (Editor-T.Sharma)- Verlag Narosa Publishing House.39-50.
8. H.S.Bhamrah (1998) Molecular Cell Biology (2nd edition). Anmol Publication Pvt. Ltd. New Delhi. 292-95
9. http://www.people.virginia.edu/arjh94/slidlist.html
10. http://www.selu.com/bio/cyto/text/banding.html
11. Masahiro Hizumo, Fukashi Shibata, Yukie Maruyama Teiji Kondo (2001). Cloning of DNA sequences localized on proximal fluorescent chromosome bands by microdissection in Pinus densiflora Sieb & Zucc. Chromosoma vol-110, issue 5, pp-345-354
12. Mohan.P.Arora (2000) Molecular Biology (First edition) Himalaya Publishing House. 262-65
13. P.K. Gupta (1999). Cytogenetics (1st edition) Rastogi Publication, Meerut.
14. Torbjorn Caspersson. (1973) Fluorometric Recognition of Chromosome and Chromosome Regions. Fluorescence techniques in Cell Biology.( Editor –A. A.Thaer & M. Sernetz) .Springer Verlag .New York. 107-23
Chromosome specific staining (Gene based)
Human Karyotype using fluoroscent staining
Q-banding - fluorescent G-banding with Quinacrine
