IN9800143

IGC-174 ^ CO 1996

Fluorescence in situ Hybridisation with reference to Biodosimetry: A Review

P Venkatachalam Solomon FD, Paul and R K Jeevanram

DERWTMENT O< ATOMIC ENERGY INDIRA GANDHI CENTRE FOR ATOMIC RESEARCH KALFAKKAM 29-11 IGC-174 1996

GOVERNMENT OF INDIA DEPARTMENT OF ATOMIC ENERGY

Fluorescence in situ Hybridisation with reference to Biodosimetry: A Review

P.Venkatachalam, Solomon F.D. Paul and R.K. Jeevanram

Indira Gandhi Centre for Atomic Research Kalpakkam 603 102, Tamil Nadu, INDIA. CONTENTS Page No.

1. Introduction 2 1.1. Chromosomal rearrangements 2 1.2. Cytogenetic techniques 2 1.2.1. Dicentric chromosome 3 1.2.2. Premature chromosome condensation 4 1.2.3. Micronuclei 5 1.2.4. Banding 6 2. Fluorescence in situ hybridization (FISH) 6 2.1. History and development 8 3. DNA probes 9 3.1. Types of DNA probes 9 3.1.1. Repeated sequence probes 9 3.1.2. Whole chromosome probes 10 3.1.3. Specific locus probes 10 3.2. Construction of DNA probes 10 3.3. Source of chromosome 11 3.4. Isolation of metaphase chromosome 12 3.5. Sorting of chromosomes 13 3.6. Cloning 15 3.6.1. Restriction enzymes 15 3.6.2. Vectors 16 4. Labelling of DNA probes 17 4.1. Direct labelling 18 4.2. Indirect labelling 18 4.3. Types of labels 19 4.3.1. In situ hybridization using radioactive labels 19 4.3.2. In situ hybridization using non-radioac tive labels 19 4.4. Labelling methods 21 4.4.1. Nick translation 21 4.4.2. Random primer elongation 22 5. Detection system 23 5.1. Detection tags 23 5.2. Fluorescence microscopes 23 6. Factors affecting in situ hybridization (ISH) 25 6.1. Slide pretreatment process 25 6.2. Target accesibility 25 6.3. Probe size and concentration 25 6.4. Hybridization rate 26 6.5. Stringency condition 27 6.6. Competitors and accelerators 27 7. Fluorescence im situ Hybridization (FISH) 28 7.1. Method 30 7.1.1. Metaphase chromosome preparation 31 7.1.2. Probe labelling 32 7.1.3. Hybridization 33 7.1.4. Post hybridization washing and detection 32 8. Application of FISH in biodosimetry 33 8.1. FISH in trans location analysis 33 8.2. FISH in dicentric chromosome scoring 36 8.3. FISH in premature chromosome condensation analysis 37 8.4. FISH in micronuclei scoring 37 8.5. FISH and banding 38 9. Conclusion 38 10. Acknowledgments 39 11. Glossary 40 12. References 43 ABSTRACT

Many advances have taken place in the field of radiation biodosimetry in the recent past. Measurement of di.centric chromosome aberrations, was first developed and followed by micronuclei scoring. These, however, are unstable type aberrations and the cells carrying such aberrations are eliminated from the body in few years. They are therefore of use primarily in case of accidental exposures. The challenge is to measure the cumulative radiation exposure resulting from normal operations by measuring stable chromosome aberrations. Banding technique can measure stable chromosome aberration but require long time to analyse the banding pattern to study translocations. On the other hand fluorescence in situ hybridization (FISH) technique is sensitive, fast and easy to identify the translocations as the chromosomes involved in translocation are painted with different colours. This review brings out the requirements of various materials, their preparations, method of detection of fluorescence etc. for carrying out FISH. The experience of various laboratories using FISH in the monitoring of radiation absorbed dose is discussed.

Key words Biodosimetry, Cytogenetic techniques, in situ hybridization, Fluorescence dyes, DNA probes, Translocation. FLUORESCENCE IN SITU HYBRIDIZATION WITH REFERENCE TO BIODOSIMETRY : A REVIEW P. Venkatachalam, Solomon F.D. Paul and R.K. Jeevanram

1. INTRODUCTION In situ hybridization is a powerful and versatile technique which allows identification and detection of nucleic acid sequences within the cells in tissue. The inherent tendency of base pairing of nucleotide sequences with the complementary sequences is the basic principle involved in this technique. This technique has good sensitivity and fine resolution in detecting and locating translocations because of the availability of fluorescent labelled DNA probes in different colours and good quality fluorescent microscopes. This review is mainly confined to application of in situ hybridization to biodosimetry.

1.1. CHROMOSOMAL REARRANGEMENTS Exposure to ionizing radiation causes chromosomal aberrations. The aberrations are of two types namely stable and unstable aberrations depending on their persistence after cell division. The number of these aberrations increase with radiation exposure and can be observed easily in human peripheral blood lymphocytes and has come to be is used as an index to measure the degree of exposure. Ionizing radiation induces breaks in chromosomes which may result in exchange of the segments. This leads to the formation of asymmetrical exchanges like dicentrics, tricentrics, rings and symmetrical exchanges like translocations or inversions (Brown, 1993). vChromosomal rearrangements

mentioned above are seen when cells are exposed either in Go or G] stage. The rearrangements will be of chromatid type if the celiS are exposed in S phase. The various types of these rearrangements and their formations are described in detail elsewhere (IAEA Technical Report: 260, 1986, Bender et al 1988; Paul etal 1996).

Health and Safety Division, Safety Research & Health Physics Group, IGCAR, Kalpakkam 1.2. CYTOGENETIC TECHNIQUES The various methods available for the detection of chromosomal rearrangements are generally known either by the type of end product looked for or by the technique used to observe the end product. Generally used methods include 1. Dicentric chromosome (DC) 2. Premature chromosome condensation (PCC) 3. Micronuclei (MN) 4. Banding and 5. Fluorescence in situ hybridization (FISH) Among these techniques, the first three are employed to detect unstable aberrations. Unstable aberrations lead to cell death when the cells undergo division. Lymphocytes carrying such unstable aberrations disappear from circulation with a biological half-life of one to two years (Bender et al, 1988). Hence, the techniques for measuring unstable aberrations are useful only for acute exposures which could occur during accidents and are measured soon after. Cumulative exposures received from low level radiation can be monitored using the last two techniques such as banding and FISH. These techniques measure stable aberrations like translocations. Stable aberrations do not lead to cell death during division and hence they remain long period. The advantages and disadvantages of these methods are summarized in Table-1.

1.2.1. DICENTRIC CHROMOSOME Scoring of dicentric chromosomes in metaphase preparation is a fairly sensitive technique for estimation of radiation dose {Lloyd et al, 1980). The nature of the dose-response curve varies with the quality of radiation and dose rate. The frequency of dicentrics increases linearly with dose for high LET radiation, and in a linear quadratic fashion for low LET radiation. This technique allows differentiation between whole and partial boa'y exposures (IAEA Technical Report:230, 1986). In whole body exposure the distribution of dicentrics among lymphocytes follows Poisson distribution and in partial body exposure they are overdispersed. The number of chromosomal aberrations for a given dose, obtained in lymphocytes are found to be the same whether the cells are exposed in vitro or in vivo. This has been observed from animal experiments and also from cancer patients exposed to whole body radiation (Natarajan, 1992).

Table-1 Cytogenetic Techniques - A Comparison

Technique S.No. Parameter CA PCC MN Banding FISH 1 Culture time 48hrs 2hrs* 72hrs ^ 48 hrs 48 hrs 2 Scoring speed (cells/day) ~ 150 -200 - 750* -25 -750* Type of Stable/ Stable / 3 aberration Unstable Unstable Unstable Unstable* Unstable* detectable Period of 4 detection after 2-3 yrs 2-3 yrs 2-3 yrs > 30 yrs* > 30 yrs* exposure 5 Quality of spread Good Good Good Good Average* 6 Base line frequency 0.001* 0.002 0.01.5 0.015 0.001 7 Sensitivity 0.10 Gy 0.05 Gy* 0.25 Gy O.lOGy O.lOGy * Advantageous compared to other tecniques

1.2.2. PREMATURE CHROMOSOME CONDENSATION This technique involves the fusion of synchronized mitotic cells to interphase cells, resulting in the premature condensation of interphase chromatin into discrete chromosomal domains called prematurely condensed chromosomes (Johnson and Rao, 1970). Initially, Sendai virus was used to fuse Chinese hamster ovary (CHO) cells and interphase cells. The problems associated with culturing and maintenance of Sendai virus were avoided by using polyethylene glycol (Panteli.as and Maillie, 1983). The factor responsible for the initiation of PCC is not well characterized. The peripheral lymphocytes are fused with CHO cells immediately after radiation exposure. The prematurely condensed chromosomes are then visualized, two hours after fusion for quantification of chromatid fragments and for studying the rearrangements. The chromosomes in eukaryotic cells are visible only at metaphase stage of the cell cycle. For this purpose the lymphocytes have to be stimulated with mitogen and cultured for two days. Within this period one would not expect all the cells to enter cell division in response to mitogen. In addition, some of the damaged cells could undergo death at interphase and some may cross first cell division, inspite of colcemid treatment to arrest the cells at metaphase stage (Pantelias and Maillie, 1984). The advantage of PCC is that radiation induced chromosomal rearrangements can be scored when cells are at interphase, and the aberrations can be studied within 2-3 hours. Moreover, the phase of the cell at the time of fusion can also be identified according to the type of aberration (Hittelman and Pollard, 1984).

1.2.3. MICRONUCLEI (MN) Scoring of MN has been proposed as a method to estimate the radiation absorbed dose. Fragments of chromosomes or whole chromosomes which fail to get incorporated into daughter nuclei during mitosis, either due to spindle poison or lack of centromere, develop into micronuclei (Schmid,1975). The dose-response relationship for radiation induced MN is well standardised (Prosser et al, 1988) MN could be expressed only in cells which have completed their first mitotic division in response to mitogen stimulation. The technique developed by Fenech (Fenech and Morley, 1985) allows scoring of MN in cells which have completed their first division, by blocking the cells at cytokinesis stage. The advantage of this technique is scoring of MN is easy and fast when compared to that of dicentrics. 1.2.4. BANDING This technique can be used to measure cumulative dose. Bands are generated on chromosomes when metaphase chromosomes are treated with enzymes like trypsin and/or heat combined with basic dyes. Banding allows the identification of individual chromosome and chromosomal regions within it and also of specific chromosome alterations like translocation, inversion etc. G-banding has been used to estimate the dose received by atomic bomb survivors (Ohtaki et al, 1982) and long term occupationally exposed radiation technologists (Kumagai, et al, 1990). Studies on the exposed radiation technologists have shown that symmetrical rearrangements increase with increase in age and dose.

2. FLUORESCENCE IN SITU HYBRIDIZATION This technique is based on the affinity among nucleotide bases in homologous sequences compared to non-homologous sequences. By using DNA probes one could selectively paint a chromosome. The DNA probes in their native form, are invisible for detection. Therefore these probes are labelled with fluorescent molecule. During hybridization the fluorescently labelled DNA binds to its complementary sequence which can be seen under fluorescence microscope. Using this technique a few chromosomes can be selectively painted with fluorescently labelled probe and if any rearrangement has taken place in these labelled chromosomes it can be seen easily. This is because the chromosomes which are not painted with fluorescent material are .stained in different colour. This can be seen in fig-1. In the photograph chromosome-4 is painted in green with fluorescent DNA probe. The metaphase spread, whe.n viewed, will show two chromosomes (pair of chromosome-4) having fluorescence and all other chromosomes in a different colour provided there is no translocation. If we consider there is a translocation between chromosome-4 and ^ay chromosome-8 then the metaphase spread will show three fluorescent chromosoi.nes. In one case FIG. 1 FITC LABELLED #4 CHROMOSOME SHOWING TRANSLOCATION the full chromosome will have the fluorescence and the remaining two will be having two different colours indicating translocation.

2.1. HISTORY AND DEVELOPMENT The development of in situ hybridization was started during 1970. Initial studies were performed with DNA probes labelled with radionuclides (Pardue et al, 1970, Jones and Gall, 1971, Henderson et al, 1972). Probes labelled with radioactive material were studied by autoradiography following hybridization. The images obtained were not sharp due to scattering of emitted radiation. Prolonged exposure of film to radiation is needed to achieve better resolution. In addition, it was necessary to count large number of metaphases to establish the results with confidence and hence there was a need to look for an alternative method. In 1980s a number of non-radioactive labels emerged which made it possible to overcome the pitfalls of radiolabelled in situ hybridization. Langer (Langer et al, 1981) was the first person who used biotin as a label for nucleic acid probe. Following this, other labels like mercury (Bauman et al, 1983), acetylaminofluorine (AAF) (Landegent et al, 1984), sulphonates (Morimoto et al, 1987) etc. were also developed for the above puquose. The labelled probes are identified by using antibodies attached with fluorescent dyes with help of fluorescence microscopes. Recent developments of in situ hybridization includes chromosomal in situ suppression hybridization (Lichter et al, 1988; Pinkel et al, 1988; Trask, 1991), multiple fluorescence in situ hybridization (Nederlof et al, 1989; 1990; 1992) and chromosome painting (Lucas et al, 1989c; 1992a; 1993). These have potential application in biological dosimetry. 3. DNA PROBES DNA probes have long nucleotide sequences and their length varies from as low as fifty to as large as thousands of bases. The probes could be either single (ssDNA) or double stranded (dsDNA) and they are classified into three types depending on their length and nature of nucleotide sequences.

3.1. Types of DNA probes 1. Repeated sequence probes, 2. Whole chromosome probes, 3. Locus specific probes (Gray et al, 1991).

3.1.1.Repeated sequence probes The chromosomes present in humans as well as in other organisms, carry certain nucleotide sequences which are repeated several times. These are called repetitive or satellite sequences, located predominantly at the heterochromatic region (Trask, 1991). For example, the centromeric region of the chromosome consist alpha satellite sequence which contains approximately 171 base pair monomeric repeats (Kameoka et al, 1990). Similarly, the telomeric region of the chromosome has beta satellite sequence which consists of 341 base pair monomeric repeats (Blackburn, 1991; Moyzis, 1991). Apart from the above, Alu and Kpn sequences are also repeated in all chromosomes (Tkachuk et al, 1991). The extent of repetition being differ at different sites within the same chromosome. They are described as "variable number of tandem repeated sequences" (VNTRS) and are unique to each and every chromosome (Kirby, 1990). Presently probes for ithese sequences are available. These probes are useful in chromosome enumeration studies (Meyne et al, 1989; 1^90). 3.1.2. Whole chromosome probes There are chromosome specific composite probes of length about 6xl06bp, which have homology sequence at many sites and this enables an entire chromosome to be hybridized. (Gray et al, 1991; Tkachuk et al, 1991). Since these probes stain the entire chromosome, they are called 'painting probes' (Pinkel et al, 1988). These probes contain certain families of repeated sequences. Since the repeated sequences are present in almost all chromosomes, there could be a cross hybridization when these probes are used for in situ hybridization. To avoid cross hybridization these sequences are suppressed by adding unlabelled competitors like human placental DNA, sonicated salmon sperm DNA (Lichter et al, 1988), or human Cot-I DNA (Ferguson-Smith, 1991). Whole chromosome probes are useful in chromosome aberration analysis (Pinkel et al, 1988; Lucas et al, 1989a; 1992a; Lucas and Straume, 1992; Natarajan et al, 1992a; 1992b; Straume etal, 1991; 1992; Schmid et al, 1992).

3.1.3. Locus specific probes In certain pathogenic conditions, a particular locus of a given chromosome is frequently involved in structural rearrangements (Cremer et al, 1988; Tkachuk et al, 1990). Once such a locus of a given disease is identified, it is easy to construct a probe for that sequence. In situ hybridization studies using these probes allows the determination of such rearrangements rapidly. Length of these prcbes vary from 50-500 bp.

3.2. Construction of DNA probes The construction of DNA probe is a difficult and time consuming process. The various steps involved in this process are given in the flow chart (fig-2). The important steps include choice of source chromosome, culture and isolation of chromosomes and cloning (Mcleod et al, 1984).

10 3.3. Sources of chromosomes Somatic cell hybrids (Hamster X Human) or human fibroblast cell lines or human lymphoblastoid cell lines can be used as a source for chromosomes. Each

FIG-2. DNA PROBE • CONSTRUCTION

CELL CULTURING CHAR0N-21A (VECTOR) 1 i ISOLATION OF CHROMOSOMES DNA EXTRACTION 4 i STAINING & FLOW SORTING RESTRICTION ENZYME DIGESTION i 1 EXTRACTION OF DNA CHARON-21A FRAGMENTS 1 i RESTRICTION ENZYME DIGESTION PHOSPHATASE TREATMENT OF ARMS

LIGATION OF ARMS & HUMAN DNA FRAGMENTS i PACKAGING OF RECOMBINANT MOLECULE IN-VITRO i INTRODUCTION OF RECOMBINANT DNA IN JO A HOST(E.COLI) i AMPLIFICATION 1 WASHING & PURIFICATION source has certain limitations. Hamster X human cell lines grow well in monolayer culture and are convenient for purification and sorting The larger

11 chromosomes can be isolated easily. However, (a) overlapping of peaks during flow karyotyping, in the case of chromosomes having lower DNA content, (b) instability of human chromosomes due to rearrangements with hamster chromosomes and (c) requirement of individual cell lines for each chromosomes, limits the use of somatic hybrids as a source in the construction of probes. The human diploid fibroblast and lymphoblastoid cells can overcome these limitations for the following reasons. It is easy to identify and isolate the desired chromosome because of their DNA content. They have stable chromosomes with out any rearrangements. They contain number of chromosomes for which isolation of more than one chromosome simultaneously during sorting (Van Dilla et al, 1986).

3.4. Isolation of metaphase chromosomes Human fibroblast cells and lymphoblastoid cells grow in monolayer and suspension culture respectively. The hamster X human hybrid lines grow in any of the cultures mentioned above. Cloning requires about 4 xlO6 chromosomes of each type with high purity. In monolayer culture, the mitotic index is in the range of 50-90%, whereas in suspension culture, it is only 10-60%. The cells are arrested at metaphase with colcemid and then given hypotonic treatment. These

chromosomes are then isolated by using either hexylene glycol buffer, MgSO4 or poiyamines to stabilize and also to yield high quality chromosomes. Distinct advantage of using the hexylene glycol buffer is that the isolated chromosomes can be identified by banding after sorting to check the purity of sorted chromosomes. Presence of large amount of cellular debris and degradation of DNA affects the utility of hexylene glycol method. Whereas, treatment with MgSO4 or poly amines yield high quality chromosomes without any degradation. The disadvantage of this raethod is that the isolated chromosomes can not be banded for cytogenetic analysis (Van den Eugh et al, 1984).

12 3.5. Sorting of chromosomes The isolated chromosomes, are stained with one or more fluorescent dyes like Hoechst-33258, Propidium iodide, DAPI, or Chromomycin-A3. The technique of staining with Fig. 3 Flow Sorting ULTRASONIC single fluorescent dye and TRANSDUCER analysis by single laser beam analyser is known as univariate karyotyping. The SAMPLE chromosomes are classified UV LASER based on their fluorescence intensity (Lucas and 458nm LASER Bartholdi, 1989). Since the fluorescence intensity is PHOTOMULTFLIERS approximately proportional to the DNA content, the r classification is also based i on the DNA content. The chromosomes having different DNA content show separate peaks, which are DISCARD easy to isolate whereas chromosomes having similar and low DNA content show cverlapping peaks which are difficult to isolate. To overcome the above limitations, methods were developed to sort and purify chromosomes based on both D.NA content and base composition. This is done by using dyes with different prefe rences for DNA of different base composition. Hoechst-33258, for example binds preferentially to AT rich region and Chromomycin-A3 to GC rich region o.f DNA. The fluorescence intensity is then measured in dual laser beam flow analyser, which

13 generate well isolated peaks even for smaller chromosomes. In addition, the centromeric index vs. DNA content and chromosome shape parameters improve chromosome discrimination (Lucas and Gray, 1987). The principle involved in flow sorting is shown in fig-3. Sheath fluid with fluorescently stained chromosome suspension is introduced into the flow chamber at high pressure. The bottom of the flow chamber has a quartz-capillary tube, where chromosome illumination and fluorescence emission occurs by the passage of UV and 458 nm laser beam. The downstream end of the quartz capillary tube has an outlet of about 75-80 |j.m diameter, through which the sheath fluid exits at very high speed. The liquid jet is broken down into droplets by piezoelectric transducer driven by high frequency oscillator (20-40 kHz). The fluorescence signal from the stained chromosome is focused onto a photomultiplier tube by lens. The light signal converted into electrical signal which is amplified and passed to charging circuitry. When a wanted chromosome (example #5) is sensed, its two fluorescence signals fall within the preset window, a charging circuit is activated which waits until the wanted chromosome is carried into the jet to form droplets. At this moment the charging circuit applies a small voltage pulse to the jet, so that when this droplet with the #5 chromosomes separates from the jet, it is electrically charged. The charging circuit remains off until another #5 chromosome sensed. Thus, when the train of droplets traverse the deflection plates, which is connected to high voltage power supply, the charged droplet containing #5 chromosomes are deflected into the collecting vessel and other droplets collected as waste. The DNA from sorted chromosomes is extracted by standard procedure (Gray et al, 1987). The number of chromosomes required to provide enough DNA for one cloning experiment was estimated to be about 106 (Griffth et al, 1984).

14 3.6. Cloning Cloning is a process where a fragment of DNA is multiplied several times. The DNA from sorted chromosomes is first cleaved into fragments with the help of restriction endonucleases, then introduced into a vector and the resultant recombinant DNA molecule is transfected into a host (example, E.coli), where multiplication takes place.

3.6.1. Restriction enzymes Restriction enzymes are endonucleases, which cleave large double stranded DNA into small fragments. There are two types of restriction endonucleases. One of these is nonspecific to site of cleavage and thus makes small random fragments from large DNA. The other enzyme is characterized by the specific cleavage site in DNA. It has a remarkable ability to cleave DNA at specific sites. These enzymes have been isolated from several strains of bacteria and are named according to the bacterium from which they are isolated. Based on the recognition site for cleavage, restriction enzymes are further divided into three types such as class I, II, and III. The recognition sequence for these enzymes are palindromic, that is, the sequences of bases at the cleavage site is identical on both strands of DNA, when read in the 5'-->3' direction. The class I and III enzymes produce a blunt end at cleavage site (the site of cleavage is at middle of the both strands) whereas, class II enzymes produce sticky ends at the cleavage site (the site of cleavage is unequal in both strands). The recognition sequence for the above class enzymes are as follows: ENZYME RECOGNITION SITE Class II 5'G*AA TTC CTT AA*G5' Class I and III 5'GG* CC CC* GG 5' * -Restriction site

15 Among these, class-II enzymes are important for cloning purposes. The recognition sequence of these enzymes varies from 4 to 6 nucleotide bases, which are palindromic in nature and the breaks produced by these enzymes are asymmetrical with sticky ends (Watson et al, 1987). Frequently used enzymes are E.coli-I and Hind-III, each having a different recognition site.

3.6.2. Vectors Vectors are extra chromosomal DNA elements like plasmids, phages, cosmids and yeast artificial chromosomes (YACs) and they have the ability of independent replication. A plasmid is a replicon (unit of genetic material capable of independent replication) that contains small circular double stranded DNA. A portion of the circular DNA, known as "replication origin", is recognized by the DNA synthetic machinery of the host, which helps the replication of the plasmid. Replication starts irrespective of the nature of DNA carried by the remainder of the plasmid. Thus a foreign DNA which can not replicate by itself, can be multiplied in this way. The ability of more rapid replication of plasmids than the bacterial host provides high degree of amplification. In. plasmids, a small DNA sequence about 10 kb size only can be inserted (Collins et al, 1991). The other vectors such as lambda phage, cosmids and YACs (Riethman et al, 1989) can accept inserts of size of 15-20 kb, 45 kb and 50-250 kb DNA respectively. The next step of cloning is ligation, in which the vector and foreign DNA is ligated together by the action of enzyme ligase. The critical factor at this stage is that the vector and the foreign DNA should be digested with the same restriction endonuclease; then only the sticky ends can get ligated perfectly. The DNA fragment produced by restriction enzyme is incubated overnight in /igation buffer

16 (0.5 M Tris-Cl, pH- 7.8, 100 raM MgCl2, 200 raM dithiothreiol, 10 mM ATP, 500|ig/ml bovine serum albumin and DNA ligase 1 unit/ml at 15 C). Once the ligation is over the cloned vector is introduced into a host bacterium by either transformation or transduction and allowed to multiply. The colonies of bacteria are plated on agar, lysed and target DNA is purified and used as a probe. The various steps in the construction of probe are depicted in fig-4. Fig. 4 DNA Probe Construction Currently DNA probes for all human chromosomes are .HUMAN GENOMIC DNA 2.FRAGMENTS OF HUMAN DNA available from American Type Culture Collection, Rockville, Maryland, USA. The o chromosomes for these 3.RESTRICTION ENZYME CUT KECONBINANT PLASMIDS PLASMID VECTOR DNA probes were sorted by flow cytometry at Lawrence Livermore National

5 RECOMBINAKT PLAS.MID 6.COLONIES OF BACTERIA CONTAINING Laboratory California INTRODUCED INTO BACTERIUM S1NCLE RECOMBINANT PLA5MIDS (E.coli digested fragments) and Los Alamos Laboratory, Los Alamos (Hind-III digested fragments) (Van Dilla and Devan, 1990).

4. LABELLING OF DNA PROBES DNA probes obtained as above are labelled either with radioactive isotopes such as 3H, 32P, or 35S, or with non-radioactive substances like fluorochromes, enzymes, biotin, digoxigenin, amino-acet.yl derivatives, sulphonates, mercury, dinitro phenyl derivatives and gold (Keirnari,1990). During labelling the label of

17 interest is artificially attached to nucleotides of the DNA. Labelling of probe is necessary because it identifies and locates the DNA probe following hybridization. Currently both direct and indirect labelling procedure are available to label DNA probes.

4.1. Direct labelling In this method the labels are directly coupled to the nucleic acid by covalent bonding. The labelled spot can be viewed under microscope immediately after hybridization. The advantages are that this is a fast method and nonspecific binding of immunochemical reaction does not occur. The disadvantage is that small target sequence can not be detected (Raap et al, 1990). Fig-5a.

4.2. Indirect labelling In this method, the DNA probes are labelled with a hapten, namely biotin, digoxigenin etc. The probes labelled with hapten are detected by immunochemical staining after ! TARGET DNA ° hybridization (Raap et al,

a Liiiiiiii»iijiuLjui___jLjmiiuji.iiuiiLi LLUIIIII FLUOROCHROME LABELLED PROBE 1990). The advantage of Fig. 5a Direct Labelling Method this method is that it allows the detection of small target DNA Fig. 5b Indirect Labelling Method . „ „, b & TARGET DNA sequences, due to signal amplification by applying D more than one layer of Q Q O Q imnr-ino - chemical B10TIN detection molecule Fig-5b. The disadvantage is it produces signal at nonspecific target region.

18 4.3. Types of labels 4.3.1. In situ hybridization using radioactive labels The stable phosphorous at either a or y position in the sugar phosphate of the DNA is replaced by radioactive 32P enzymatically. Nucleotides labelled with 35S contain phosphorothioate group (P=S) instead of phosphate group (P=O) at either a or y position. Tritium is another radionuclide used in the form of tritiated thymidine for labelling DNA probe. Among these 32P is commonly used because of its availability with various specific activities and its high energy which is useful for quick detection. The disadvantage is it has short half-life and poor resolution to distinguish closely located targets because of its high energy. In the case of 35S it has longer half-life and lower energy which provide high resolution, but sensitivity is low due to position modification during labelling and low energy. Tritium has long-half life, provides a low background but require longer exposure time to get good resolution (Cunnigham and Mundy, 1987). Though radiolabelled probes are still being used, they have certain limitations like cross hybridization due to non-specific binding, handling and safety cost of disposing of waste materials (Me Neil, 1991).

4.3.2. In situ hybridization using non-radioactive labels To overcome the limitations of radiolabelled DNA probes, non-radioactive labelled probes are being introduced for in situ hybridization. Among non- radioactive labels, fluorochromes are incorporated directly into the probe and detected without immunochemical staining. The three types of fluorochromes available now are fluorescein isothiocyanate (FITC), t>°tramethyl rhodamine isothiocyanate (TRITC), and aminomethyl coumarine acetic acid (AMCA) (Yoshida, 1992). Fluorochromes are cationic dyes introduced ;nto the DNA by a transient opening of helix without unwinding, a mechanism known as

19 intercalation. The anionic phosphate group in the nucleic acid is also capable of binding with fluorochromes. The staining properties of fluorochromes is strongly influenced by pH, because during staining ion exchange processes take place, in which small cations from the target are competitively displaced by larger cations of dye. When the dye and target are together for a longer period, then there is an equilibrium which results in optimum intensity during excitation (Keirnan, 1989). Biotin and digoxigenin are used as hapten, in indirect labelling. Biotin is a vitamin derived from metabolic products of many micro-organisms. The major function of biotin is acting as a coenzyme or prosthetic group in several catalytic events. During biotinylation, it gets attached to different positions in nucleotides. The significant property of biotin is that during labelling it forms a very tight complex with avidin, the detection tag. Digoxigenin, a protein derived from Foxglove plant, produces less background interference compared to biotin. The favorable site of attachment of biotin or digoxigenin in nucleotides are (Fig-6) C-5 position of cytosin and uracil, C-6 position of thymine, C-8 position of Fig. 6 Site for Biotin attachment in various nucleotides CYTOSINE URACIL

THYMINE

GUANINE 9^m ADENINE

CARBON 9 OXYGEN IN NITROGE;EN 'O j»°J ATTACHMENT HYDROGEN

20 guanine and adenine, those positions which are not involved in hydrogen bonding. Rarely N-4 position of cytosine and N-6 position of adenine get biotin attachment (Keller and Manak, 1989).

4.4. Labelling methods The label either radioactive or non-radioactive gets incorporated into the probe either by nick translation or random primer elongation.

4.4.1. Nick Translation Various steps are involved in this technique, as shown in fig-7. In the first

Fig. 7 Nick Translation

NICK V p p p ^ p p p p ?

* O^ 8 g

Jddddddd 5

1.DOUBLE STRAND DNA 2 NiCK PRODUCED BY RESTRICTION ENDONUCLEASE

NICK

5 p p p \ J r r T SUGAR

BASE Q

3.DNA POLYMERASE-I REMOVES THE 4.DNA POLYMERASE-I REPLACES THE NUCLE0T1DE AT THE NICK MISSING NUCLEOTIDE WITH A LABELED NUC'LEOViDE step, enzyme DNase-I induces a nick on one of the strands of DNA probe (dsDNA) in a random manner, to create a free 3'-OH group. Next, enzyme 5'->3' exonuclease removes one or more bases at 5' side of the nick. The gap is then filled by labelled nucleotides, available in the medium by the acuon of DNA polymerase-I at the 3' termini. Like this the cycle is repeated till the ^rid of the

21 probe length. Finally, the probe is purified to remove enzymes, unincorporated label and salt to reduce non-specific background noise. This is carried out by either ethanol precipitation, gel filtration or hydrophobic chromatography. Nick translation is an easy, rapid and relatively inexpensive procedure for producing uniformly labelled probes (Rigby et al, 1977). This can label both circular and linear dsDNA but not single stranded DNA. Under optimal conditions 60-70% of labelled nucleotides present in medium can be incorporated. 4.4.2. Random primer elongation Fig. 8 Random Primer elongation Random primer 5' DOUBLE STRAND DNA 3. elongation, is an alternative 3' 5' method for labelling of probes. DENATURATION This is carried out by using 1 3' 3' primers, developed by 3' Fienberg (Fienberg and Vogestein, 1984). In this IPRIMER ADDITION technique dsDNA is denatured 5' by means of physical treatment 3' '5' to get ssDNA. Single stranded DNA obtained is then mixed LABELLED dNTPs IIPRIMER ELONGATION with hexanucleotide primers of 5' 3' random sequence. This results 3' in the hybridization of primers at multiple sites along the DENATURATION 1 PURIFICATION ssDNA. The enzyme Klenow LABELLED DNA DNA polymerase elongates the primer at 3'-OH termini by using labelled nucleotides present in the medium. Finally, they are purified by any of the methods mentioned in nick translation and used for in situ hybridization experiments (Fig-8).

22 This method is applicable only to label small targets of linear ssDNA and dsDNA but not circular DNA. At present, several companies are supplying reagents in the form of kits for nick translation and random primer elongation procedure (Lewis, 1994) for convenient labelling.

5. DETECTION SYSTEM 5.1. Detection tags Various methods are available to detect the probe hybridized with the target. The radiolabelled probes are detected by autoradiography on an x-ray film. Radiation emission from the labelled probe interacts with the silver halide, present on the film emulsion and reduces it to silver. This forms a latent image, visible on processing the film. The time required to expose x-ray film to get good resolution of hybridized probe depends on the radioisotope used as a label and the same has been explained under section 4.3.1.. The probes labelled with non-radioactive substances are detected either by colorimetric method or by fluorescence microscope depending on the type of label used. The DNA probes labelled indirectly with hapten are detected either by streptavidin and enzyme conjugated antibodies or fluorescein avidin conjugated antibodies. The most commonly used enzymes for the detection of hybridized probes are horse-radish peroxidase (Papdimitride et al, 1976) and alkaline phosphatase (Hopmann et al, 1988). The alkaline phosphatase reaction depends on the production of the insoluble formesan from nitroblue tetrasodium chloride, which is seen best under phase contrast optics (Garson et al, 1987). Whereas the reaction of peroxidase depends on the oxidation of diaminobenzidine which produces a dark brown insoluble complex, which appears as a bright spot at the hybridization site (Landegent et al 1985). The biotin or digoxigenin labelled probes are detected either by fluorescein avidin, tetrameth/! rhodamine avidin or aminomethyl coumarine avidin followed by biotinylated or antidigoxigenin

23 antibodies. Under appropriate excitation, they emit fluorescence signals visualized with the help of fluorescence microscopy. Currently several antibodies are available for the detection of hapten labelled probes (Yoshida, 1992). The hybridization spot is directly visualized under fluorescence microscopy by using appropriate filter set in the case of probes directly labelled with fluorochromes like FITC, TRITC and AMCA. The filter set used for detection of different fluorochromes are given in Table-2. Table-2 Filters used in FISH detection

S.No. Dye Colour Filter Excitation (nm) Emission (nm) 1 FTTC Green 490 560 2 TRITC Red 530 580 3 AMCA Blue 335 400 4 PI Red 546 590 5 DAPI Blue 340 420

5.2. Fluorescence microscopes The developments in fluorescence microscopy also play an important role in the detection system. Earlier, the probes labelled with single fluorochrome hybridized with target DNA were detected by using dual band filters, Later, detection of dual colour was achieved by changing the filter set and dichroic mirror followed by double or triple exposure without shifting the object (Nederlof et al, 1990). With this technique, it is not possible to visualise closely spaced probes due to image shift. Presently, the development of double band filter set with wide range of wavelength allows the detection of double colour simultaneously (Lucas et al, 1993). Exact registration of images using CCD device (Hiraoka et al, 1987), computerized digital image processing analysis (Arndt-Jovin et al, 1985) and storage based on fluorescence intensity ratio, opens the gateway for multiple fluorescence in situ hybridization (Nederlof et al, 1992; Ried et al, 1992; Wiegant et al, 1992).

24 6. FACTORS AFFECTING IN SITU HYBRIDIZATION Since in situ hybridization is a multistep procedure, successful result depends upon various factors. This includes preservation of target sequences, pretreatment of slides, accessibility of the target to probe, probe size, complexity and concentration, high efficiency of hybridization with reduced background signal, stringency condition, and choice of competitors and accelerators (Keller and Manak, 1989; Harries and Wilkinson, 1990; Me Neil et al, 1991). These factors are described in following paragraphs. Failure of any of these parameters can result in a lower signal to noise and loss of sensitivity. 6.1. Slide pretreatment processes Aging of slides containing metaphase chromosomes or tissue preparations plays a role in the results of in situ hybridization. A maximum of 24 hour period is recommended at room temperature for the aging of slides and about six months in the nitrogen atmosphere (Drafler et al, 1992). The slides stored in nitrogen have to be removed and kept at 65°C for four hours prior to process. In case of slides having tissue sections the slides should be treated with RNase for a period of one hour at 37°C followed by proteinase K treatment to prevent cross hybridization with endogenous ribonucleic acid and protein. (Pinkel et al, 1986). However, such treatment is not required for the slides containing chromosome preparations. 6.2. Target accessibility The factor which is most critical for the success of in situ hybridization is the accessibility of the target DNA for high molecular weight DNA probes (Raap et al, 1990). This can be achieved by pretreatment of slides with RNase and proteinase-K as mentioned above in case of tissue preparation. 6.3. Probe size and concentration The size of the probe, depends on the nature of \hv study. The whole chromosome composite probes, provide good signal because they have several

25 complementary sites along the entire length of the target. Even though whole chromosome probes are derived from several clones, because of the size of the clone, complexity and high molecular weight, the target accessibility is somewhat difficult. This can be rectified by selecting large number of clones with small insert sequences of bases. On the other hand, small locus specific probes may be considered which have easy accessibility to the target. The problem with locus specific probe is the signal strength because of the short length of the probe. This however, can be overcome by signal amplification (Scherthan et al, 1992). High concentration of probe increases the efficiency of signal detection, and hybridization rate but affects the hybridization efficiency. Within narrow limits, sensitivity increases with increasing probe concentration. The concentration limit is not determined by any inherent physical properties of the probe but by the type of label and detection reagent used. 6.4. Hybridization rate Hybridization rate is the time required for reassociation of denatured target and probe, which depends on the length of the probe and its concentration, temperature, ionic strength, viscosity and pH (Keller and Manak 1989). The time required to hybridize half of the probe to its target sequence can be calculated by using the formula (Meinkoth and Whale, 1984).

t1/2 =log2/KC (1) Where K = rate constant for hybrid formation (Mol/liter/nucleotides) C = probe concentration in solution (moles of probe molecules/liter) The rate constant K-depends upon probe length (L), probe complexity (N, ratio of G:C), temperature, ionic strength, viscosity and pH. For example, a probe length of 40-monomer that contains two copies of a 20 nucleotide sequence, L=40 and N=20. The relationship of K to these variables is: 05 K = KnL /N (2)

26 5 + Where Kn is the nucleation constant and is 3.5 x 10 for Na concentrations of 0.4 - 1.0M, pH values of 5-9 and hybridization temperatures 25°C below the Tm of probe-target hybrids. To calculate the rate, in seconds, for hybridization of half of the probe to its target, equations (1) and (2) can be combined to give: 5 5 ti/2 = Nlog2/3.5xl0 (L°- )C For a probe of 500 bases in length, the value of ti/2 would be: 5 10 tl/2 = 500 (0.693) / 3.5xl0 (22) 6xlO" = 75,000 seconds (20 hours) It is found that the probe length and hybridization half time rate is proportional to the probe length. So, longer the probe length, hybridization half time ia also longer leads to reduced hybridization efficiency. 6.5. Stringency conditions The stringency of the hybridization conditions determines the hybrid stability which depends predominantly on melting temperature (Tm). Tm is the melting temperature, at which half of the DNA is denatured to form single stranded DNA. This is influenced by probe complexity, length, salt and formamide concentration. For highly complex probes (i.e., high GC base composition) which are long, the Tm is high and they form stable hybrids. Higher concentration of monovalent cations, especially sodium increases the stringency and the stability of hybrid. Formamide decreases the melting temperature. Generally, hybridization occurs 25°C below the melting temperature. To achieve this formamide is used. Therefore by altering any of the above parameters, we can achieve the required stringency condition and stability of the hybrid and in turn the sensitivity. 6.6. Competitors and acclerators Using whole chromosome probes especially in non-radioactive hybridization good sensitivity is not achieved due to low signal- noise ratio i.e., due to non-specific hybridization (Pinkel et al, 1988); LicMer et al, 1990). The required staining contrast is achieved by using unlabelled genomic DNA in

27 hybridization to block non-specific binding. Sonicated salmon sperm DNA, human placental DNA and Cot-1 DNA are successfully used to prevent the interaction between non-specific target and fluorescently labelled probe (Lucas et al, 1992a). The cot-I DNA consists of mainly repetitive sequences, will renature fast when compared to unique nucleotide sequences during hybridization. Since the concentration of such sequences are more in cot-I DNA than the repetitive sequences present in the probe, it will competitively bind with other chromosomes and prevent non specific hybridization. Though the cot-I DNA have the chance to hybridize with the chromosome of our interest, it could be selectively prevented by the probe which we have used. It is because the probe contains more homologous sequence to the target chromosome than cot-I DNA. Highly complex probes need longer time for hybridization which in turn reduces the hybridization efficiency, and chances for self reannealing is high. To eliminate these draw backs accelerators like inert polymers, dextran sulphate, polyethylene glycol, poly acrylic acid, phenol and guanidine thiocynate are used to accelerate hybridization rate. The acclerators mediate by lowering viscosity of the hybridization buffer and the energy difference between target DNA and probe.

7. FLUORESCENCE IN SITU HYBRIDIZATION Fluorescence in situ hybridization (FISH) is a technique in which DNA probe is labelled with fluorescent molecule for easy identification and detection of nucleic acid sequence within a cell or a tissue preparation. Fluorescence is a process in which light energy of one wavelength is absorbed by the fluorescent material and then released after a short time (billionth of a second) as light energy of slightly longer wavelength. This is because there is some energy loss during absorption and emission. The resulting difference in wavelength is called Stokes shift. This wave length shift is vital because the wave length of the light used to excite the fluorescent molecules can be very precisely chosen and the light

28 emitted from the molecule can be very precisely observed and discriminated from the excitation light. Fig-9 illustrates the fluorescence phenomenon. The peak on the left is the light energy that can excite the fluorescent molecule and the peak on Fig. 9 Excitation and Emission Spectra 1 00

EXCITATION BANDPASS

20 --

0 - 300 500 600 700 LIGHT WAVELENGTH (.itm) the right is the light energy emitted by the molecule. Generally, fluorescent compounds or fluorochromes, have characteristic colour of absorption and emission. Quinine, for example absorbs ultraviolet light and fluoresces with blue light emission, while fluorescein absorbs blue light and fluoresces with yellowish green light. As mentioned earlier Stokes shitt plays a,a important role in fluorescence detection. The factor that is determining the sensitivity is the relative intensity of the signal to that of background (noise). The sensitivity of fluorescence detection is determined by 1. the ability to separate fluorescent light from exc iting light. 2. the ability to detect very low light intensity and 3 the presence of interfering fluorescence known as 'autofluorescence". In order to separate fluorescent light from the exciting light, filters are available. Fluorescence filters are designed to transmit v ery specific wavelength of light while excluding all others. The most versatile fluorescence filters are

29 interference filters which work by using fluorescence characteristics of wavelengths. One type of filter is called a band pass filter because it allows a "band" of wavelengths to pass through. Band pass filters can be made to pass very narrow spectral bands of light which provides superior specificity. However, very small bands naturally transmit lesser amount of light, are harder to see, and thus decrease sensitivity. Typically, a band pass filter is chosen wide enough for the application to collect as much light as possible but not so wide as to create spillover into other bands of interest. The emission and excitation band pass filters must be so chosen that they transmit two separate wavelengths. Fig-10 shows that filter A (excitation filter) passes light band of wavelength 35nm wide and centered at 535nm at the middle. Filter B (emission filter) passes a light band of wave length 35nm wide and 590nm at the middle. The light seen through filter B is from the probe, because the illuminating light is not visible through filter B. Fig. 10 Filters for illumination and visualisation of labelled probes

WIDE SPECTRUM SAMPLE WITH LIGHT SOURCE LABELLED PROBE FILTER-A FILTER-B

DETECTION BY EYE (OR) „ CAMERA O- HA

535 EXCITATION PASS f>90 EMISSION PASS

7.1. Method FISH technique involves four major steps: 1. Preparation of metaphase chromosome 2. Labelling of DNA probes 3. Hybridization 4. Post hybridization washing and detection.

30 7.1.1. Preparation of Metaphase chromosome About 0.5ml heparinised peripheral blood is cultured in a suitable medium containing growth hormone. Phytohemagglutinin (PHA) is added to the above culture to make the lymphocytes enter the cell cycle. Cultures are incubated at 37°C. Colcemid is added to the culture to arrest the lymphocytes at metaphase 3 hours before the end of the culture. At the end of 48 hours cells are harvested, treated with hypotonic solution(0.45% KCl) for 20 minutes) and fixed in Carnoys fixative (methanol and acetic acid in 3:1 ratio). After three washes with the fixative, the cells are cast on a slide by dropping the cell suspension from a height of 10 cm. 7.1.2. Probe labelling Depending on the nature of study, the probe is selected and labelled with the method described under section 4.4. The probes are then purified on sephadex G-50 column to remove the unlabelled nucleotides and enzymes (Fan et al, 1990). 7.1.3. Hybridization The events involved in hybridization are depicted in fig-11. Prior to Fig. 11 Visualisation with the in situ hybridization

Target DNA Labelled Probe

Denatured Denatured target DNA labelled Probe

Hybridization / Hybrid DNA \AAA n/w

31 hybridization the slides with metaphase chromosomes are denatured at 70°C for 2 minutes in 70% formamide. The slides are then dehydrated and cooled in ethanol series of 70%, 80%, 90% and 100% for 3 minutes each and air dried. Similarly DNA probe, suspended in hybridization buffer composed of 50% formamide/ 2X SSC/ 10% dextran sulphate and human placental DNA is denatured by heating it for 5 minutes at 70 C. The above denatured probe is allowed to stand for one hour at room temperature before applying over the prewarmed slides containing metaphase chromosome. This is covered with a coverslip, sealed with rubber cement and hybridization is carried out at 37°C for 16 hours (Pinkel et al, 1986). Prehybridization of denatured probe for one hour is not required if human cot-DNA is used instead of genomic placental DNA in the hybridization buffer (Lichter and Cremer,1992). 7.1.4. Post hybridization washing and detection In case of probes labelled with fluorochromes, the coverslip is removed Fig. 12a Detection system Fig. 12b Detection system (Directly labelled probe) (Indirectly labelled probe)

FLUORESCENT CONJUGATED AVIDIN Ab FLUORESCEIN ISOTHIOCYANATE

v-**"'

0 ii *f \m

K-l (-CH

H.C'v Jc»—ICN,].

\S COOK s

BIOTIM

ADCNiNt

CYT01 « A TUtUWt THYU1NC

OEOXYSIlWSE SUGAR • PHO«>BATE BKOUf ^HOSPHATC CRQU*

32 after 16 hour hybridization and the slide rinsed three times in 50% formamide/ 2 X SSC, 2 X SSC for 10 minutes and once in 2 X SSC/ 0.1% NP-40 for 5 minutes at 45°C. After air drying the slides are counterstained with DAPI or PI (Drafler et al, 1992). For indirectly labelled probes, after overnight hybridization, the coverslip is removed and the slides are washed three times, with 50% formamide/ 2 X SSC/at pH 7, once with 2 X SSC and once in PN buffer (mixture of 0.1 M NaH2Po4 ,

0.1M Na2HPo4 and 0.1% Nonidet Po4) for 10 minutes each at 42-45°C. An additional wash in PN buffer for 10 minutes at room temperature is recommended. The bound probe is detected by alternating incubation in avidin-FITC and biotinylated goat avidin antibody both in PNM buffer (PN buffer 5% nonfat drymilk 0.02% sodium azide). First the coverslip is removed and the slides are immersed with PNM buffer containing avidin-FITC at 37°C for 20 minutes. Then the slides are washed three times in PNM buffer for three minutes each. Finally the slides are incubated with antiavidin antibodies for 20 minutes at room temperature. For amplifying the signal the above steps are repeated. Finally, the slides are counterstained, mounted and viewed under microscope. The principles behind this method are shown in fig- 12a and 12b.

8. APPLICATION OF FISH IN BIODOSIMETRY 8.1. Application of FISH in translocation analysis Pinkel (Pinkel et al, 1986), first applied this technique to study radiation induced chromosome rearrangements in human X hamster hybrid cell lines. Since then several authors reported the dose dependent increase of translocation and its persistence several years after exposure (Bouffler et al, 1992; Lucas et al, 1989a, 1989c, 1992a, 1992b; 1993; Lucas and Straume, 1992a, 1992b; Gray et al, 1992, 1994; Nakano et al, 1993; Natarajan et al, 1992; Schmid et al, 1992; Straume et al, 1992; Bauchinger et al, 1993; Matsuka ei al, 1993; Tucker et al, 1993).

33 Lucas et al (Lucas et al, 1989a) have used locus specific probe and reported that the dose response curve for dicentric chromosome and translocation have same pattern. In addition, they also reported that translocation frequency detected by FISH is higher compared to G-banding. The reason attributed for this is the difference in scoring of aberration. Translocation studies using whole chromosome probes was successfully utilized to study the exposure of (a) victims of Goiania accident (Straume et al, 1991), (b) an occupational worker (Straume et al, 1992) and (c) a person accidentally exposed to tritium (Lucas et al, 1992b). The above studies clearly indicate the persistence of stable aberrations long time after exposure. Estimation of dose in sample exposed in vitro have shown that radiation induced translocations are higher than the dicentrics by a factor ranging from 1.2 to 9(Natarajan et al, 1992a, 1992b; Schmid et al, 1992; Nakano et al, 1993; Matsuka et al, 1994). The possible reason is the variation in scoring ability; i.e. some dicentrics might be scored as translocations (Straume and Lucas, 1993). The two colour hybridization technique was developed, to locate exact position of centromere in the entirely painted chromosome (Van Denken, 1988; Meyne et al, 1989). FISH technique using whole chromosome probes for chromosome 1, 2, 4 and centromeric probes for all the chromosomes was performed to estimate dose in Hiroshima A-bomb survivors (Lucas et al, 1992a). This method allowed detection of rearrangements involved in the entire target chromosomes and permitted to distinguish dicentric and translocation. The studies confirmed that stable aberration detected by G-banding and FISH are not statistically different. Using FISH, a limited number of translocations only can be detected as the number of chromosomes painted are few in number. In order to obtain the entire genomic translocation frequency a mathematical formula is used (Lucas et al, 1989b).

34 Fg = Fp / 2.05 fjp (1-fp) Where Fg = Total genomic transiocation frequency Fp = Transiocation Table-3 DNA Content in Human Chromosomes Chromosome DNA content frequency detected by Number (% of Autosome Total) FISH 1 4.32 2 4.22 fp = Fraction of 3 3.49 4 3.34 genome painted 5 3.20 In the above 6 3.02 7 2.77 formula fraction of X 2.70 genome painted is 8 2.55 9 2.38 determined based on its 10 2.37 DNA content. The DNA 11 2.35 12 2.33 content of all the human 13 1.86 chromosomes is given in 14 1.80 15 1.69 Table-3 (Mendelsohn et 16 1.55 al, 1973). The formula 17 1.55 18 1.49 derived is based on the 20 1.40 assumption that 19 1.20 Y 0.92 1. Radiation induced 22 0.86 breakpoints are 21 0.82 distributed randomly throughout the genome. 2. Hot spots on certain chromosomes may not be an important contributor to the overall transiocation frequency. 3. Probability of a chromosome being involved in the transiocation is dependent on its DNA content (Lucas et al, 1992). The relationship between DNA content and its translation frequency can be calculated by using the equation:

35 Fi = 0.0058 + 1.81di Where, Fi = Translocation frequency di = DNA content of a particular chromosome The equation clearly shows that the translocation frequency increases with increase in DNA content. Natarajan et al, have shown high translocation frequency in chromosome 1 compared to chromosome 2, 3,4, 8, and X (Natarajan et al, 1992b). As mentioned earlier, the detection efficiency increases with the fraction of genome painted. In single colour FISH, the efficiency with which the translocation frequency changes in a parabolic manner as a function of fp, i.e. the fraction of genome painted. In double colour FISH, a maximum of 50% translocation can be detected. Using three colour FISH the detection efficiency can be increased upto 67% (Lucas and Straume, 1993). The procedures of multiple FISH is not yet applied for dosimetric studies. However, if part of a chromosome or the entire chromosome or more than one pair is painted, the total genomic translocation frequency remains the same. 8.2. Application of FISH in dicentric chromosome scoring Dicentric chromosomes are scored conventionally by staining with Giemsa, for measuring radiation absorbed dose. At times pairs of touching chromosomes or chromosome with twist may appear like dicentric chromosome. It will be difficult to analyse the spread with homogeneously stained chromosome preparations. One can over come these problems using FISH technique. Using locus specific probe Lucas et al (Lucas et al., 198S0 have constructed dose response curve for both dicentric chromosome and translocation frequency. Thus FISH technique has certain advantages over Giemsa metf'od in scoring dicentric chromosomes.

36 8.3. Application of FISH in premature chromosome condensation analysis Brown (Brown and Evans, 1992) found a solution to the difficulties involved in the scoring of aberration in PCC by combining it with FISH. They used human diploid fibroblast cell lines (AG 1522) fused to Hela cells to induce PCC. After preparing the slides, whole chromosome probe for chromosome 4 was hybridized and analyzed for the rearrangements and its repair kinetics. From that they found, the number of breaks produced in the single chromosome and rate of rejoining as a function of time after irradiation in a single chromosome is representative of breaks and rejoining of whole genome. The authors have reported translocation detected by FISH in metaphase cell and rearrangements detected by PCC in interphase cells are comparable for the same dose. (Brown et al, 1992). 8.4. Application of FISH in micronuclei (MN) studies Though cytochalasin-B technique allows to differentiate cells which have completed their first division from rest, it is difficult to tell whether the MN is derived from fragments or whole chromosome. Using FISH technique it is possible to get the above information and also one can find which chromosome is frequently involved in MN formation. It would be useful to find out if there are any 'hot spots" in the genome, for radiation induced chromosomal rearrangements. In 1992 Miller (Miller et al, 1992), used FISH Co analyse chromosome composition of MN and its numerical distribution. They used biotin labelled centromenc and telomeric probes for chromosomes and the labels used were detected by avidin-FITC and AMCA antigens and its Antibodies. If the MN has both centromeric and telomeric signals of the probe then it is assumed to be derived from whole chromosome; if MN showed signal only for telomeric probe then it is derived from acentric fragment and if it has more than 2 signals, it is a fusion of fragments. Thus FISH is useful in knowing the origin of MN formation.

37 8.5. Application of FISH in banding studies G-banding, though useful to quantify long term exposure cases, has certain limitations like requirement of good metaphase spreads and experience for analysis of banding pattern. Giemsa trypsin (GTG) banding combined with FISH can overcome these limitations (Lichter and Cremer, 1992). Popp and Cremer demonstrated the feasibility to have a cytogenetic dosimeter by combining GTG banding with FISH (Popp and Cremer, 1992). G- banded slides with metaphase chromosomes were used for in situ suppression hybridization and counterstained with DAPI. As a result the entire chromosome appears in three different colours. Because GTG banding is performed prior to CISS-hybridization, it allow conventional chromosome banding analysis in addition to the analysis of painted chromosomes. This technique permits the detection of translocation occurring between chromosome of different colured subsets and unequivocal identification of ring chromosomes, chromosome fragments, and micronuclei.

9. CONCLUSION FISH technique is a fast and sensitive technique to analyse stable aberrations, mainly translocation. With the help of this one can measure cumulative radiation exposures in occupational workers. Certain difficulties involved in analysing dicentric chromosomes, premature chromosome condensation and banding could be overcome by this technique. In addition to biodosimetry, FISH technique has potential application in various fields of biology, such as genome mapping and enumeration studies, gene expression studies, clinical genetics, prenatal diagnosis, microbiology, virology, somatic cell analysis and meiotic studies (Lichter and Ward, 1990). The reasons for the application of FISH ir various fields are given below: 1. Good spatial resolution, speed, sensitivity and the stability of the probe.

38 2. Probes can be modified with variety of reporter molecules both chemically and enzymatically. 3. Detection system permits simultaneous visualization of differentially labelled probes in different colours. 4. DNA sequence can be identified in metaphase chromosomes and in interphase nucleus. 5. The entire genome of a particular species, entire chromosome,chromosomal sub regions or single copy sequences can be specifically highlighted by using complex probes. 6. Enhanced resolution and sensitivity by improvement in digital imaging microscopy and amplification system respectively. 7. Suppression of cross hybridization by using unlabelled genomic DNA or human cot-I DNA as a competitor. 8. Easy to automate the technique.

10. ACKNOWLEDG MENTS The authors sincerely express their gratitude to Shri L.V.Krishnan, Director, Safety Research and Health Physics Group and Shri A.R.Sundararajan, Head, Health and Safety Division, for their constant encouragement and valuable suggestions extended during the preparation of this report. The authors also wish to thank Dr.P'iazid Rodriguez, Director, Indira Gandhi Centre for Atomic Research for his keen interest in the research activities in biological sciences. The authors also grateful to UGC for providing finacial support.

39 11. GLOSSARY Alpha satellite sequence A repeated sequence of nucleotides in DNA molecule at the centromeric region of all chromosomes. Alu sequence A family of closely related dispersed sequence of 300 nucleotides, many of which contain a common cleavage site for the restriction enzyme Alu I. Avidin A protein (mw 68000 daltons) present in the white of raw hen's egg. A molecule of avidin consists of four identical subunits and can bind noncovalently to four molecules of biotin. Avidin antibody Antibodies to avidin raised in animals like horse, rabbit, goat etc. Avidin FITC Avidin conjucated to a fluorescent dye, the fluorescein iso thio cyanate. Beta satellite sequence DNA consists of repeated sequence of nucleotides at telomeric regions of all chromosomes. Biotin A vitamin which acts as a cofactor in carboxylation and transcarboxylation reactions. It is also used as a tag in nucleotide labelling for in situ hybridization. Cosmid A plasmid into which has been inserted the cos site of bacteriophage lambda. Cosmids can be used as vectors for cloning large DNA fragments in the construction of genomic libraries.

40 Cot-1 DNA Short, simple or highly repeated sequences will quickly renature followed by denaturation. The product of concentration of nucleotides and time of reaction is known as cot unit. It may be expressed in terms of moles of nucleotides per seconds per liter. Cross hybridization Binding of a probe to a DNA sequence other than the intented target sequence. This occurs because of homology between the sequence and low stringency hybridization wash conditions are followed. Hapten A substance which can elicit an immunoresponse only when combined with other molecule; if administered in its own it fails to stimulate antibody production. Hot spots Some regions in a chromosome mutate much more frequently than other regions. Hybridization Formation of double stranded nucleic acid by base pairing between single stranted nucleic acid derived from different sources. Lymphoblast cells Cells present in lymphoid tissues, which are organised in the form of organs such as lymph node, spleen, thymus and aggregation at gastrointestinal tract. They are responsible for the production of lymphocytes. Monolayer culture A population of cells is incubated in a shallow layer of growth medium until the cells form a more or less confluent layer of one cell k'hick, on the inner surface of the culture vial.

41 Nick translation A process in which "DNA polymerase one" simultaneously polemerises new DNA and degrades DNA ahead of the growing site, that can continue some distance beyond the intial primer end. Stringency contltions The buffer salt concentration and temperature used in posthybridization wash process. As. these parameters are changed, the degree of binding of probe to target DNA changes. Vector Any living organisim which effects the transmission of cloned DNA from one individua 1 to another. Yeast artifi cial chromosome A vector used in gene cloning. It contain both centromere and telomere which function as stable chromosome ends, allows to replicate as small linear chro/mosome.

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