New methods in cytogenetics

New methods in cytogenetics

VJ Buckle and 1 Kearney

Introduction

Institute of Molecular Medicine, Oxford, UK

Developments in the technique of fluorescence in situ hybridization (FISH) now permit hybridization of sequences ranging from 1 kb to whole genomes. The technique can be used in applications from coarse mapping of whole chromosomes to high-resolution analysis of extended strands of DNA. The complexity, and hence the coverage, of ‘paints’ prepared by amplification is being improved to the extent that such methods are used in cloning strategies for the generation of region-specific probes. Interphase analysis and comparative genomic hybridization are becoming important tools in cancer cytogenetics, and the potential for routine analysis of fetal cells obtained from maternal blood may provide a fresh approach to prenatal cytogenetic screening. Functional studies of gene activity and nuclear organization are

now also possible.

Current Opinion in Genetics and Development 1994, 4:374-382

The field of molecular cytogenetics has been revolu- tionized in recent years by advances in the technique of fluorescence fn situ hybridization (FISH), which have provided powerful new tools for investigating chromosome structure and function. These developments include new haptens and fluorochromes, combinatorial and ratio labelling for multicolour signals, new equipment for the detection of such signals and novel hybridization targets. The largest improvements have been in probe generation - an enormous range of material can now be used as the hybridization probe, principally through the use of DNA amplification. In this review, we discuss the developments in detail and place them in the context of their applications within genetic research and clinical diagnosis.

Generating probes

The type of material that can now be used readily as hybridization probe has expanded rapidly, ranging from small sequences of genomic DNA or cDNA through whole-chromosome ‘paints’ to the whole genomes used in comparative hybridization. Paints are now commercially available for all human chromosomes, as are probes for most centromeres, some telomeres and a range of specific loci. The principal advance in probe generation has been the ability to amplify selected DNA by PCR and to incorporate la-

bels directly into the amplification reaction, with suitable primers selected according to the application.

Chromosome paints Whole-chromosome or region-specific paints can be produced by interspersed repetitive sequence/element (IRS/IRE) PCR, which relies on species-specific primers for consensus sequences in human Ah or Kpn repeats. These primers will selectively amplify the human content in somatic cell hybrids and yeast artihcial chromosome (YAC) clones and will generate paints from flow- sorted chromosomes, although such paints suffer from the uneven distribution, spacing and orientation of repeat elements within the genome. New approaches to overcoming such problems include the use of dual Ah primers of opposite orientation 111 and ligation of an anchor oligonucleotide to digested DNA (IRE- bubble PCR) 129 which improves the complexity of the amplified DNA. Chromosome-specific libraries prepared by linker-adaptor PCR also produce a more even paint 13’1. The technique of degenerate oligonucleotide primer (DOP) PCR on flow-sorted chromosomes pro- duces complex DNA and an even paint 141 and is not species specific, so can be used to amplify DNA from any source. One method by which to rapidly character- ize de nom abnormalities is to flow sort the abnormal chromosome, amplify and label, and then hybridize back to normal chromosomes. This reverse chromosome painting uses small numbers (200-500) of flow- sorted chromosomes to create a paint for the abnormal chromosome in order to directly identify its constituent parts El.

374

Abbreviations CGH-comparative genomic hybridization; DOP-degenerate oligonucleotide primer; FISH--fluorescence in situ hybridization;

IRS/IRE-interspersed repetitive sequence/element; PCR-polymerase chain reaction; PRINS-primed in situ labelling; YAC-yeast artificial chromosome.

0 Current Biology Ltd ISSN 0959-437X

New methods in cytogenetics Buckle and Kearney 375

Multicolour hybridizations An increasing number of spectrally separable fluorochromes are available and multiple fractions of the chromosome complement can now be distinguished simultaneously by means of combinatorial labelling 161 or ratio labelling 171 in which, currently, 12 targets can be discerned by ratio-coding with different proportions of three fluorochromes. The availability of directly fluorochrome-conjugated nucleotides for labelling probes provides additional variety and an efficient alternative to immunocytochemical detection WI. The ability to visualize multiple colours in multiplex ,hybriditations with paints and YACs has led to the concept of ‘chromosomal bar codes’, specific patterns of differentially coloured chromosomes, with the aim of constructing sets of probes tailored to specific diagnostic needs [!Pl. A combination of chromosome paints and YACs or cosmids can also be used as an alternative to reverse painting, where marker chromosomes cannot be separated by flow sorting 1101.

Microdissection Region-specific probes can be generated for any part of the human genome by microdissection of human chromosomes with direct enzymatic amplification and labelling of the microdissected DNA fragments for use as hybridization probe 111,121, thereby avoiding time-con- suming microcloning procedures. A modification using topoisomerase I to relax supercoiled DNA has allowed the generation of probe from a single microdissected chromosome, reducing the risk of contamination with DNA from other chromosomes 113’1. Microdissection in combination with FISH has been used to examine chromosome abnormalities associated with malignant melanoma 111,14*1 and chronic myeloid leukaemia 1151 and to define the derivation of homogeneously staining regions [16] and double-minute chromosomes 1171.

Primed in situ labelling Primed in situ labelling (PRINS) 1181 involves the an- nealing of an oligonucleotide primer to chromosomal DNA followed by labelling and in situ extension. DNA thermal cyclers with a flat-plate hotblock now allow fixed chromosomes on microscope slides to undergo a series of cycles similar to PCR, termed cycling PRINS. A drawback to repeated denaturing over a large number of cycles is the diffusion of the labelled product away from the site of hybridization, causing inaccu- rate localization 119’1. Although sensitive enough to allow the detection of low copy number sequences on metaphase cells 1201, the application of cycling PRINS to the detection of short, single-copy sequences remains to be determined. One report to date has described the successful localization of a single-copy gene by PRINS 1211, although the success was dependent on enhancement by digital imaging microscopy.

Comparative genomic hybridization One of the most significant new strategies in the analysis of chromosomes in tumours is one which re- quires no culture or metaphase preparation from the tumour material and no prior knowledge of the chromosome constitution. Comparative genomic hybridization (CGH) provides a novel way of determining differences in copy number between test (turnour) and reference (normal) DNA 1221 (see Fig. 1). The fluorescent signals are quantitated using a digital image analysis system, with a software program to calculate the ratio of red to green fluorescence along the length of each chromosome. Some limitations and pitfalls must be considered, however. CGH cannot detect balanced chromosome rearrangements, no information is available on the way gains and losses are arranged within marker chromosomes and the presence of normal tissue within tumour tissue is a potential problem. It is important to carry out normal/normal hybridization controls for each CGH experiment to determine aver- age fluorescence ratios for the normal chromosomes, as considerable overlap is found between normal and test genomes 123”l. Quantitation of fluorescence val- ues may be more reliable using directly fluorochrome- labelled probes, rather than immunocytochemical detection.

CGH appears to be more sensitive for the detection of amplifications than for deletions, although the limits of sensitivity have yet to be determined. The problems of heterogeneity in tumour samples may be overcome by the use of DOP-PCR amplification of selected areas of tumour tissue followed by CGH analysis. It has been demonstrated recently that this approach is feasible on archival solid tumour samples 1241. A recent modification of reverse painting uses whole tumour genomic DNA as a probe 1251, which, like CGH, results in an increase in fluorescence where there is an over-representation of sequences, and a decrease where there is deletion.

Most of the publications concerning CGH to date have described the investigation of copy number in solid tumours. Increasing evidence, however, points to it being a useful technique for the analysis of leukaemic samples, particularly in lymphoid neoplasias. CGH analysis of acute myeloid leukaemias and chronic lymphocytic leukaemias has been reported recently 1261. The major- ity of chronic lymphocytic leukaemia cases showed significant differences between the results from CGH and G-band analysis. Confirmation of the abnormalities by interphase FISH indicates that the population of cells stimulated for cytogenetic analysis may not be repre- sentative of the clonal changes in chronic lymphocytic leukaemia.

Hybridization targets

Extended DNA Signals from differentially labelled probes can be re- solved by FISH on chromosomes when separated by

376 Genetics of disease

Normal metaphase spread of human chomosomes

Fig. 1. A schematic representation of comparative genomic hybridization (CCH) analysis. Differentially labelled tumour DNA (shaded) and normal DNA

IU I I (white) are hybridized to a normal meta- Normal Amplication Deletion phase spread of human chromosomes.

Regions of gain of DNA sequences are

n seen as an increased fluorescence in-

Fluorescence tensity from the tumour probe, whereas

ormal DNA signal losses of DNA sequences result in a pre- dominantly normal hybridization signal. The fluorescence intensities along the

Tumour DNA signal medial axis of the chromosome are determined using a digital image analysis system. The ratio of fluorescence inten-

Tumour:normal sity gives a quantitative estimate of the copy-number variations in the tumour. Unlabelled Cot-l DNA included in the hybridization blocks the binding of la-

0 1994 Currenl Opinion in Genetics and Developmenl belled DNA at the centromeric repeat areas (striped). Adapted from 1701.

l-2Mb of DNA. Below this figure, mapping informa- Interphase analysis tion can be obtained by looking at the arrangement of probe signals in Gl interphase nuclei, in which the chromatin is less condensed; sequences can best be ordered at interphase in the 50-500 kb range. Re- cent techniques have increased the resolution of FISH interphase mapping by the preparation of free, linearly extended DNA. Wiegant ef al. 1271 released DNA by de- tergent treatment to form loops (halos) around the nuclear matrix. The advance of the direct visual hybridization (DIRVISH) technique 128V lies in providing a stream of linear DNA strands across the slide. The extended DNA allows work at a fine resolution and the feasibility of multiplex hybridizations, combining small fragments with YACs or cosmids, means that fragments can be localized within a larger clone. Clones can be oriented and ordered by examination of only a few images, and contigs can be characterized with gaps and overlaps defined. The technique also provides good sensitivity, with strings of signal from genomic fragment probes of under 5 kb, although the beaded appearance of the signal produced by hybridizations to extended DNA 127,28”,29*1 does warrant some con- sideration [SOI. An advantage of the halo preparations is the ability to identify signals from both homologues around a nucleus 131**1. The loops of halo preparations are thought to represent the chromatin lying between nuclear attachment sites and this may permit the comparison of the distribution of active and inactive genes with respect to the nuclear scaffold.

The use of interphase nuclei as hybridization targets for the examination of chromosome aneuploidy or rearrangement bypasses the need for a dividing cell population and enables screening of large numbers of cells. This has provided access to a variety of novel tissues, including individual agar colonies of haemopoietic progenitor cells 1321, destained and archival blood smears 1331 and paraffin-embedded tissue sections 134,351.

Several studies have emphasized the value of interphase FISH analysis for the detection of numerical abnormalities in neoplastic cells. Chromosome-specific repetitive probes have been used to detect mono somies and trisomies in haematological malignancies [33,36-381 and provide a way to assess whether single cells with an abnormal karyotype represent an emerging clone 1391. Studies of chronic lymphocytic leukaemias have demonstrated that the frequency of trisomy 12 137,401 and deletion of the retinoblastoma (RI311 and p53 genes [40,41”1 was greater than determined by G-band analysis. The detection of mono- somies has a false positive rate of lO-20% and may fail to detect partial loss of chromosomes caused by translocation 1361. Careful design of the controls as well as evaluation of the data is extremely important, espe- cially if these procedures are to be used for minimal residual disease detection.

Interphase FISH has been used for the detection of residual disease after allogeneic bone marrow trans-

New methods in cytogenetics Buckle and Kearney

Fig. 2. Examples of fluorescence in sifu hybridization (FISH) with a variety of probes and targets. (a) Reverse painting on normal chromosomes with a probe derived by DOP-PCR amplification of a flow-sorted de nova derivative chromosome 16. The paint highlights both of the normal chromosomes 16 and the terminal portion of the short arm of chromosome 9, from which the extra material on the de nova chromosome 16 derives. Chromosome 9 is identified by an additional centromere-specific probe. (b) An extended DNA preparation hybridized with two overlapping cosmid probes from 16~13.3 which have been differentially labelled with biotin (red signal) or digoxigenin (green signal). The region of overlap appears yellow. (c)and (d) Identification of the (4;ll )(q21;q23) translocation which occurs in acute leukaemia. (c) A YAC containing the breakpoint region within llq23 detects the translocation in leukaemic chromosomes. Fluorescent signal is observed on the normal chromosome 11 (arrow), the translocation chromosome 11 (closed arrowhead) and the translocation chromosome 4 (open arrowhead). (d) Cosmids flanking the 1 lq23 breakpoint detect the translocation in leukaemic interphase cells. The differentially labelled cosmids give one juxtaposed red and green signal per nucleus, representing the normal chromosome 11, and two discrete red and green signals where they have been separated onto different translocation chromosomes. The nuclei are counter-stained with DAPI (blue). Images were obtained with an MRC 600 confocal microscope (a-c) or a cooled charge-coupled device (CCD) Photometrics camera, enhanced using Cenejoin software (d). Acknowledgement to Gaby Senger for cd).

plantation 1421. Ried ef al. 143’1 have recently investigated the usefulness of interphase FISH with locus- specific probes for the detection of the (2;8)(q32;q24) translocation in artificial mixtures of normal and Burkitt’s lymphoma cells and showed that this system was very specific for the detection of the t(2;8), with only 0.01% of false positive cells. Long-term studies are necessary to determine the level at which residual cells herald relapse. The possibility of linking these studies to the immunophenotype of residual cells may be a better indicator of prognosis.

FISH analysis and immunophenotype can now be combined to provide three-colour analysis of immunophenotype as well as two different numerical aberrations in the same cell [44*1. These methods provide a tool for characterizing cell populations within a heterogeneous mixture, such as found in solid tumours. Simultaneous immunophenotyping and FISH analysis has been used to investigate lineage involvement in myelodysplastic and myeloproliferative syndromes 145,461. Concurrent immunophenotype and FISH analysis has also been used to demonstrate that a leukaemia which emerged


five years after sex-mismatched allogeneic bone marrow transplant occurred in donor cells 1471. So far, only numerical abnormalities have been investigated using repetitive centromere probes, but the technique may be extended to include translocations using locus-spe- c2ifl.c probes.

Digital imaging microscopy

Roth the sensitivity and flexibility of FISH techniques have been improved by the introduction of cooled charge-coupled device. (CCD) cameras. These devices are extremely sensitive to photons and exhibit an al- most perfect linear response to light. The exquisite sensitivity allows the detection of very small sequences and will be particularly useful for extended DNA analysis. The devices also have a wide dynamic range, allowing multicolour imaging, with the number of fluorochromes detectable limited only by the number of fluorescent filter sets available on the microscope. Dig- ital imaging microscopy also introduces the possibility of quantifying in sifu hybridization signals. Nederlof et al. [481 optimized the instrumentation for quantitation of fluorescent signals, but were still hampered by significant variation in the size of signals. Wxzlling with directly fluorochrome-labelled dNTPs has been demonstrated recently to be applicable to the detection of single-copy sequences and subject to less back- ground l8.1.

Physical mapping and cloning strategies

Multicolour FISH has become a rapid and flexible technique for the construction and validation of coarse and high-resolution chromosome maps and for the characterization of chromosome abnormalities. A variety of cloning strategies have developed from FISH technology as an approach to positional cloning. Coincidence painting is an extension of reverse painting methodology for cloning region-specific DNA sequences 149’1. Microclones derived from microdissection can be used to screen cosmid libraries and are an appropriate size for sequence analysis to generate sequence-tagged sites, which can then be used to screen YAC libraries 1121. Microdissection has been used recently as part of a protocol to obtain cDNAs from any selected region of the genome (50’1. Libraries prepared by linker-adaptor PCR 13’1 or by IRE-bubble PCR 12”l are also suitable for the generation of sequence-tagged sites which can be targeted to specific chromosome regions. The genomes of other species are equally amenable to analysis by the techniques described in this review 12”,511.

Prenatal and diagnostic cytogenetics

Recent advances in FISH technology have had a considerable impact on clinical cytogenetic analysis be- cause of the commercial availability of probes, the speed with which the techniques can be applied to large numbers of cells and the potential for automa- tion. FISH kits are now available for the diagnosis of common microdeletion syndromes, and both paints and region-specific probes are being used to char- acterize structural chromosome rearrangements. The applicability of interphase FISH as a screening tool for the detection of aneuploidy in uncultured amniocytes has been investigated in a large series of 4500 specimens [52**1. The study showed that 82% of all chromosome abnormalities detected by routine cytogenetic analysis. were detected by interphase FISH. This type of assay cannot readily detect mosaicism, structural abnormalities or rare aneuploidies, however, and is currently supplemented by conventional cytogenetic analysis. There may be a case for its use as a rapid screen in pregnancies at high risk for a chromosomal aneuploidy. Coelemic fluid derived from extra-embry- onic mesoderm and aspirated at 8-10 weeks has also been used for prenatal FISH [531 and may be a safer approach than puncturing the amniotic membrane. An important development is the potential for using FISH as a non-invasive screen for aneuploidy in fetal cells enriched from maternal blood [54,55**1. Sex determi- nation of pre-implantation embryos in couples at risk for X-linked disease 1561 is another potential application.

Chromosomal behaviour and function

Meiosis FISH has provided an additional tool with which to look at aspects of meiosis. The alignment of home logous sequences prior to formation of the synap- tonemal complex has been described in yeast 1571 and the behaviour of human reciprocal translocation chromosomes has been examined through first and second metaphase 1581, allowing a prediction of expected normal, balanced and unbalanced sperm.

Mechanisms of chromosome aberration Early events in the development of chromosome ab normalities can now be examined with FISH technology. The use of two-colour fluorescence at the dihydrofolate reductase locus permitted an analysis of early amplification events in Chinese hamster ovary cells and demonstrated that a giant inverted duplication was a frequent initiating event which led to breakage/fusion/bridge cycles mediating subsequent amplification 159’1. Chromosome painting has also been used to study the mechanisms by which chromosome dam-

New methods in cytogenetics Buckle and Kearney 379

age is induced by ionizing radiation 1601; this approach could be extended to other clastogens.

Nuclear organization Accumulating evidence suggests that nuclear function is closely related to three-dimensional structure and FISH has proved an essential tool for the examination of spatial organization within the nucleus. The distribu- tions of telomeres, centromeres and chromosome-specific sub-satellite domains in lymphocyte nuclei have been shown to be cell cycle dependent [611. The higher level organization of transcription, splicing and trans- port involving several RNA classes is being studied with a combination of fluorescence hybridization, immuno- fluorescence and digital imaging microscopy [62-641.

Replication timing and gene activity One important aspect of chromosomal function is that of replication. Active genes are thought to replicate early in S phase, whereas inactive genes frequently replicate later, although the generality of this hypothe- sis has not been widely tested. A gene in Gl or early S interphase nuclei probed by FISH will normally exhibit a discrete signal, whereas a doublet signal is observed after replication, representing hybridization sites on both chromatids. Selig et al. [651 used these observa- tions to determine the replication status of a number of different genes and also to define the boundaries of replication time zones for the cystic fibrosis trans- membrane regulator gene. The assay has since been used to examine the replication status for a number of mouse genes known to be imprinted and human genes for which imprinting has been implicated t661. The technique has also been used to demonstrate a dif- ference in replication timing of the fragile-X FMRl gene in normal and FRAX4 males carrying an expanded al- lele [67J, and in the identification of a specific origin of replication for the human p globin domain [68**1.

Conclusions

Cytogenetic analysis is, at present, undergoing one of the most significant advances since the development of banding techniques. With the aid of new molecular cytogenetic techniques incorporating FISH, it is now possible to accurately define karyotypes on the basis of a specific molecular reaction and not simply the sub- jective identification of chromosome bands. Technical advances over the past year have seen the wider availability of directly fluorochrome-labelled probes, as well as an increase in the number of laboratories using sensitive digital imaging devices. The sensitivity of FISH mapping techniques is now down to 1 kb 1691, and ratio imaging with four distinct fluorochromes should soon permit the discrimination of all 24 human chromosomes in one hybridization, although in most situ-

ations this facility would not be critical. Chromosome paints and region-specific probes are proving invalu- able for cytogenetic characterization in gene mapping, as an aid to positional cloning and in clinical diagnosis, and immunophenotyping and CGH promise to open up previously intractable areas of cancer cytogenetic research. Research workers are now also in a position to address important aspects of chromosome organization and behaviour.

References and recommended reading

Papers of particular interest, published within the annual period of review, have been highlighted as: . of special interest . . of outstanding interest

1. Liu P, Sicilian0 J, Scong D, Craig J, Zhao Y, de Jong P, Siclllano MJ: Dual Alu Polymerasc Chain Reaction Priicrs and Conditions for Isolation of Human Chromosome Paiit- ing Probes from Hybrid Cells. Cancer &net Cyfogenell993, 65:93-99.

2. . .

Munroc DJ, Haas M, Brie E, Whitton T, Aburatanl H, Hunter K, Ward D, Housrnan DE: IRE-Bubble PCR: a Rapid Method for Efficient and Rcprcscntativc Amplification of Human Gc- nomic DNA Scqucnccs from Complex Sources. Genomrcs 1994, 19:50&514.

This paper describes interspersed repetitive element (IRE) PCR. a new method for amplifying the human DNA content of hybrids, YAC, cosmid and phage clones, which results in greater complexity than standard interspersed repetitive sequence (IRS) PCR, and which is suitable both for FISH mapping and for the generation of targeted sequence-tagged sites.

3. Vooijs M, Yu L-C, Tkachuk D, Pinkel D, Johnson D, . Gray JW: Libraries for Each Human Chromosome, Con-

structed from Sorter-Enriched Chromosomes by Using Linker-Adaptor PCR. Am J Hum Genet 1993, 52:%36-597.

Describes the generation of complex wholechromosome libraries by digestion of flow-sorted chromosomes with a frequent cutter and ligation at each end of the resulting fragments with an adaptor oligonucleotide ready for subsequent amplification.

4.

5.

6.

Telenius H, Pelmear AH, Tunnacliffe A, Caner NP, Behmel A, Ferguson-Smith MA, Nordenskjold M, Pfragner R, Ponder BAJ: Cytogcnetic Analysis by Chromosome Painting Using Degcncratc Oligonucleotidc-Primed-Polymcrasc Chain Reac- tion Amplified Flow-Sorted Chromosomes. Genes Cbromo- som Cancer 1992. 4~257-263.

Rack K. Harris PC, MacCarthy AM, Boone R, Raynham H, McKinley M, Fitchett M, Towe CM, Lindenbaum RE, Buckle Vl: Characterization of Three de Nwo Dcrivativc Chrome somcs 16 by ‘Rcvcrsc Chromosome Painting’ and MolccuLar Analysis. Am J Hum Genet 1993, 52~387-997.

Ried T, Baldini A. Rand TC, Ward DC: Simultaneous Vi- sualization of Seven Diicrcnt DNA Probes by In Slhc Hybridization Using Combinatorial Fluorcscencc and Dig- ital Imaging Microscopy. J+vc Nat1 Acad Scf USA 1992, 89:lm1392.

Dauwerse JG, Wiegant J, Raap AK, Brcuning MH, van Om- men GJB: Multiple Colors by Fluorcsccncc in Sftu Hybridixa- tion Using RatioLabcllcd DNA Probes Crcatc a Molecular Karyotype. Hum Mol Genet 1992, 1:593-598.

Wiegant J, Wiesmeijcr CC, Hoovers JMN, Schuuring E, d’Azao A, Vrolijk J, Tanke HJ, Raap AK: Multiple and Scnsi- tivc Fluorcsccncc In S&n Hybridization with Rhodaminc, Fh~oresccin-, and Coumarin-Labclcd DNA.% Cyfogener Cell Genet 1993, 63:73-76.


The autho; tested the sensitivity of new red, green and blue fluorescent dUTPs in hybridizations. Tetramethylrt,mdamine-dUTP was most sensitive, fluorescein4UTP was rather less sensitive and coumadn-dUTP was successful only .for highly repetitive target sequences. Three-colour hybridizations were achieved by using an in- direct biotin label detected with avidiiumarin.

9. . Lengauer C, Speicher MR, Popp S, Jauch A, Tanawaki M, . . Nagaraja R, Reithman HC, Don&Keller H, D’Urso M, Sch-

less@ger D, Cremer T: Chromosomal Bar Codes Produced by Multicolor Fluorescence In Situ Hybridization with Mul- tiple YAC Clones and Whole Chromosome Painting Probes. Hum Mol Cener 1993, 2505-512.

Describes multiplex hybridizations with YAC clones and wholechromosome paints to produce multicolour bar codes, which can be de- signed to suit any analytical requirement.

10.

11.

12.

13. .

Popp S, Jauch A, Schindler D, Speicher MR, Lengauer C, Donis-Kel1er.H. Rlethman HC, Cremer T: A Strategy for the Characterization of Minute Chromosome Rearrangements Using Multiple Colot Fluorescence In SIrU Hybridization with ChromosomcSpectic DNA Libraries and YAC Clones. Hum GeneI 1993, 92:527-532.

Meltzer PS, Guan X-Y, Burgess A, Trent JM: Rapid Gener- ation of Region Specific Probes by Chromosome Microdii section and Their Application. Nature Gkner 1992, 132428.

Guan XY, Meltxer PS, Cao J, Trent JM: Rapid Generation of Region-Specilic Genomic Clones by Chromosome Microdis section: Isolation of DNA from a Region Frequently Deleted in Malignant Melanoma. Genomfcs 1992, 14&O&34.

Guan X-Y, Trent JM, Meltzer PS: Generation of Band-Specific Painting Probes from a Single Microdissected Chromosome. Hum Mel Gener 1993, 2:1117-1121.

Thls paper describes a modification of the technique for obtaining region-specific probes by chromosome midissection, using topoisomerase I prior to amplification. This improved the sensitivity of the technique, allowing the generation of a chromosome-painting probe from a single mlcrodiied chromosome.

14. Meltzer PS, Guan X-Y, Trent J: Tclomere Capture Stabilizes Chromosome Breakage. Nanrre Cenel 1993, 4:252-255.

;his study used microdissection and FISH to demonstrate that sus- pected terminal deletions of 6q in malignant melanoma were, in fact, unbalanced tmnslocations. A different donor chromosome was in- volved in each case.

15.

16.

17.

18.

19. .

Zhang J, Meltzer P, Jenkins R, Guan X-Y, Trent J: Appllca- tion of Chromosome Mlcmdissection Probes for Elucidation of BCR-ABL Fusion and Variant Philadelphia Chromosome Translocations in Chronic Myelogenous Leukemia. Bhcd 1993, 81:33653371.

Zhang J, Trent JM, Meltzer PS: Rapid Isolation and Char- acterization of Amplitied DNA by Chromosome Microd& section: Identification of IGFZR Amplitication in Malignant Melanoma. Oncogene 1993, 8:2827-2831.

Sen S, Sen P, Mulac-Jericevic B, Zhou H, Pirrotta V, Stass SA: Microdiicted Double-Minute DNA Detects Variable Patterns of Chromosomal Localizations and Multiple Abun- dantly Fbqmsd Transcripts in Normal and Leukemic Cells. Genomks 1994, 19542-551.

Koch JE, Kolvma S, Petersen KB, Gregersen N, J3olund L: OllgonuckotidePriming Methods for the Chromosome-Spe citk LabeBing of Alpha Satellite DNA In S&u. Cbromaroma 1989, 98:259-265.

Komminoth P, Long AA: In Sftu polymcrasc Chain Reaction. Vidwws Arch DJ 1993, 64:67-73.

Thls is a very good review of methodology, applications and pitfalls of k slhr PCR/cyclii primed In sffu labelling (PRINS) from a histo- &em&y perspeaive. Most of the applications to date have been in localizatiofi of viral genomes in tissue sections, and the experience of using cycling PRINS ln these situations has highlighted the potential dmwbacks of using the technique for metaphase and interphase maPPine*

20. Gosden J, Hanratty D: PCR In S&u: a Rapid Alternative to In S&u Hybridization for Mapping Short, Low Copy Number Sequences. Biotecbnkpres 1993, 15:78-80.

21. Cinti C, Santi S, Maraldi NM: Localization of Single Copy Gene by PRINS Technique. Nt~clefc Acids Res 1993, 21:579+5800.

22. Kallioniemi A, Kallioniemi OP, Sudar D, Hutovitz D, Gray JW, Waldman F, Pinkel D: Comparative Genomic Hybridiza- tion for Molecular Cytogenetic Analysis of Solid hours. Science 1992, 258:818-821.

23. du Manoir S, Speicher MR, Joos S, Schrock E, Popp S, . . Dohner H, Kovacs G, Robert-Nicoud M, Lichter P, Cramer

T: Detection of Complete and Partial Chromosome Gains and Losses by Comparative Genomic In S&u Hybridization. Hum Gkner 1993, 90~5-10.

This is an excellent investigation of comparative genomic hybridization methods for the detection of chromosome imbalances and the paper details very thoroughly the advantages, limitations and potential applications of this new technology. The importance of optimiza- tion of chromosome preparations, hybridization conditions, image acquisition and analysis are all discussed in detail.

24.

25.

26.

27.

28. . .

Speicher MR, du Manoir S. S&rock E, Holtgreve-Grez H, Schoell B, Lengauer C, Cremer T, l&d T: Molecular Cytogc- netic Analysis of Formalin-Fiicd, Pa&in-Embcddcd Solid ti mours by Comparative Genomic Hybridization after Univer- sal DNA-Amplfication. Hum Mol Genel 1993, 2:1907-1914.

Joos S, Scherthan H, Speicher MR, Schlegel J, Cremer T, Lichter P: Detection of Ampliticd DNA Sequences by Rc- verse Chromosome Painting Using Gcnomic amour DNA as Probe. Hum G&met 1993, 584-589.

Bentz M, Huck K, du Manoir S, Joos S, Schiitz B, Dijhner H, Lichter P: Identification of Previously Undetected Chrome somal Abnormalities in Leukcmias by Comparative Genomic Hybridization. Blood 1993, 82(suppl):122.

Wiegant J, Kalle W, Mullenders L, Brookes S, Hoovers JMN, Dauwerse JG, van Ommen GJB, Raap AK: High-Resolution In Situ Hybridization Using DNA Halo Preparations. Hum Mol G&wet 1992, 1587-591.

Pan-a I, Wiidle B: High Resolution Visual Mapping of Stretched DNA by Fluorescent Hybridization. Narirre C&et 1993, 5:17-21. -. This paper describes the preparation of streams of linearly extended

DNA. Cosmid signals appear as strings of beads and can be cohy- brkiiid with genomic end fragments for orientation. The authors mapped an amplification array and reported successful hybridization over the length of a 700 kb YAC.

29. Fidlerovd H, Senger G, Kost M, Sanseau P, Sheer D: mo . Simple Procedures for Releasing Chromatin from Routinely

Fiicd Cells for Fluorescence In Sftu Hybridization. Cytogenet Cell Gizner 1994, 65~203205.

This paper describes two methods for releasing chromatin from routinely harvested and fixed cells. NaOH treatment results in the complete disruption of nuclei and extended DNA strands, whereas formamide treatment maintains nuclear borders, allowing homologous signals to be examined as with halo preparations.

30. Buckle VJ, Kearney L: Untwirling Dih. Nature Genet 1993, 5:45.

31. Tocharoentanaphol C, Cremer M, S&r&k E, Blonden L, . . Killan K, Cremer T, Rkd T: Multicolor Fluorescence In S&u

Hybridization on Mctaphase Chromosomes and Interphase Hal@Preparations Using Cosmid and YAC Clones for the Siiultaneous High Resolution Mapping of Deletions in the Dystrophin Gene. Hum G@net 1994, 93:229-235.

The authors describe a multicolour FISH analysis of deletions in the dystrophin gene. Cosmids specihc for deletion-prone regions were considered preferable to YAC clones in thii context. The paper ex- plores the use of halo preparations for high-resolution analysis of small deletions, by comparison of signals on homologous chromosomes.

New methods in cytogenetics Buckle and Kearney 381 .

32. Poddighe PJ, van der Lely N, Vooijs CP, de Witte T, Ha- maekers FCS, Hopman AHN: Interphase Cytogenetics on Agar Cultures: a Novel Approach to Determine Chrome somal Aberrations in Hematopoietic Progenitor Cells. Fxp Hematol 1993, 21:859-863.

Bentz M, Schroder M, Herz M, Stilgenbauer S, Lichter P, Dohner H: Detection of Trisomy 8 on Blood Smears Us ing Fluorescence In SIIu Hybridization. Lettkemla 1993, 7:752-757.

Micale MA, Sanford JS, Powell IJ, Sakr WA, Wolman SR: Dclining the Extent and Nature of Cytogcnctic Events in Prostatic Adcnocarcinoma: Paraffin FISH vs Mctaphasc Analysis. Cancer Gener <Progener 1993, 69:7-12.

Kii SY, Kee JS, Ro JY, Gay ML, Hong WK, Hittelman WN: Intcrphasc Cytogcnctics in Paraffin Sections of Lung Tumors .by Non-isotopic In Situ Hybridization. Am J Puthol 1993, 142307-317.

Zhao L, van Oort J, Cork A, Liang JC: Comparison Bc- twecn Intcrphasc and Mctaphasc Cytogcnctics in Detecting Chromosome 7 Dcfccts in Hematological Ncoplasias. Am / Hematol 1993, 43:20%211.

Anastasi J, Le haI MM, Vardiman JW, Fernald AA, Larson RA, Rowley JD: Detection of Trisomy 12 in Chronic Lym- phocytic Leukemia by Fluorescence in S&u Hybridization to Interphase Cclis: A Simple and Scnsitivc Method. Blood 1992, 79:1796-1801. Kibbelaar RE, Mulder JWR, van Kamp H, Fibbe WE, Wes- sels JW, Beverstock CC, Hdak HL, Kluin PM: Dctcction of Monosomy 7 and Trisomy 8 in Mycloid Ncoplasia: A Com- parison of Banding and Fluorescence in Sffu Hybridization. Blood 1993, 82:904-913.

Chen Z, Morgan R, Stone JF, Sandberg AA: A Usc- ful Technique in the Vcrihcation of Clonality of Random Chromosome Abnormalities. Cancer Gener Cytogener 1993, 66:73-74.

Dohner H, Fischer K, Cabot GP, Hansen K, Pilz T, Diehl D, Stilgenbauer S, Benner A, Bentz M: lncidencc and Prognos tic Signiiicancc of Recurring Chromosome Abnormalities in Chronic ECcll Lcukemias. An Interphase Cytogcnctic Anal- ysis. Blood 1993, 82(suppl):2236. Stilgenbauer S. Dohner H, Bulgay-Monschel M, Weitz S, Bentz M, Lichter P: High Frequency of Monoallelic Rctinoblastoma Gene Deletion in BCell Chronic Lymphoid Leukemia Shown by Intcrphasc Cy-togcnctics. Blood 1993. 81:21182124.

This very elegant study used interphase FISH with a )r phage contig for the retinoblastoma gene to detect deletions of this locus in B- cell chronic lymphoid leukaemia cells. An internal control and dualcolour hybridization were used to increase the sensitivity of the assay for the detection of deletions.

42. Wessman M. Popp S, Ruutu T. Volin L, Cremer T, Knuu- tila S: Detection of Residual Host Cells after Boric Marrow Transplantation Using Non-Isotopic In SIIu Hybridization and Karyoty~ Analysis. Bone Marrow Transplant 1993, 11:279-284.

43. Ried T, Lengauer C, Lipp M, Fischer C, Cremer T, Ward . DC: Evaluation of the Utility of Intcrphasc Cytogcnctics to

Dctcct Residual Cells with a Malignant Genotype in Mixed Cell Populations: a Budtitt Lymphoma Model. DNA Celf Biol 1993, 12:637-643.

This is an evaluation of the sensitivity and specificity of interphase FISH for the detection of specific translocations in small numbers of ceils. A complex four-colour stmtegy was used to provide a very specific pattern in male Burkitt’s lymphornd cells, and the use of a CCD camera increased the number of scorable cells.

44. Weber-Matthiesen K, Deerberg J, Muller-Hermelink A, . Schlegelberger B, Grote W: Rapid lmmunophcnotypic Char-

acterization of Chromosomally Aberrant Cells by the New FICI’ION Method. cyrogenef CeN Genef 1993, 63:123-125.

This paper describes the use of combined immunophenotyping and dual-colour FISH to demonstrate cytogenetic variability within the CD30-positive Reed Sternberg cells of a Hodgkin’s lymphoma. The authors used Cy3, FITC and AMCA fluorochromes. Cy3, in place of Texas Red, was found to increase the sensitivity of immunophenotyping. 45.

46.

47.

48.

49. .

Price CM, Kanfer EJ, Colman SM, Westwood N, Barret AJ, Greaves MF: Simultaneous Gcnotypic and Immune phenotypic Analysis of lnterphasc Cells Using DualColour Fluorcsccnce: a Demonstration of Lincagc Involvcmcnt in Polycythacmia Vera. Blood 1992, 80:103~1038.

Nylund SJ, Verbeek W, Duramendy ML, Ruutu T, Heinonen K, Hallman H, Knuutila S: CeU Lineage lnvolvcment in Four Patients with Myclodyspiastic Syndrome and t(1;7) or Tri- somy 8 Studied by Simultaneous lmmunophcnotyping and Fluorcsccncc In Situ Hybridization. Cancer G&et Cytogener 1993, 70:120-124.

Katz F, Reeves BR, Alexander S, Kearney I, Chessells J: Lcukacmia Arising in Donor CcUs Following Ailogeneic Boric Marrow Transplantation for p Thalassacmia Dcmonstratcd by Immunological, DNA and Cytogcnetic Analysis. Br] Haemaf 1993, 85326331.

Nederlof PM, van der Flier S, Verwoerd NP, Vrolijk J, Raap AK, Tanke HJ: Quantification of Fluorcsccncc in Situ Hy- bridization Signals by Image Cytometry. Cyfomerry 1992, 13846052.

Bailey DMD, Carter NP, de Vos D, Leversha MA, Perry- man MT, Ferguson-Smith MA: Coincidence Painting: a Rapid Method for Cloning Region Specific DNA Scquenccs. Nn- cleft Acids Res 1993, 21:5117-5123.

This paper describes a strategy for the generation of region-specific sequences by hybridizing together chromosomes which overlap in the region of interest and then cloning the hybridized DNA. Probes derived from the cloning system are suitable for the genemtion of sequence-tagged sites and for YAC and cosmid library screening.

50. Hozier J, Graham R, Westfall T, Siebert P, Davis L: Prcpara- . tivc In S&u Hybridization: Sclcction of Chromosome Rcgion-

Specific Libraries on Mitotic Chromosomes. Genomfcs 1994, 19:441-i47.

Describes a procedure for the isolation of cDNAs or genomic DNA from specific regions of the genome using a combination of amplification, hybridization and microdissection.

51. Milan D, Yerle M, Schmitz A, Chaput B, Vaiman M, Frelat G, Gellin J: A PCR-Based Method to Amplify DNA with Random Primers: Determining the Chromosomal Content of Porcine Flow-Karyotypc Peaks by Chromosome Painting. Cyfogener Cell Gener 1993. 62:13+141.

52. Ward BE, Gersen SL. Carelli MP, McGuire NM, Dackowski . . WR. Weinstein M, Sandlin C, Warren R, Klinger KW: Rapid

Prenatal Diagnosis of Chromosomal Ancuploidics by Fluorcs ccncc In S&u Hybridization: CUnical Expcricncc with 4500 Spccimcns. Am J Hum &net 1993, 52:854-865.

A large study of interphase cytogenetics as a screening tool on uncultured amniocytes. Region-specific probes for chromosomes 13, 18, 21, X and Y were used, and this probe set detected 82% of the abnormalities reported by conventional cytogenetic analysis.

53. Jurkovic D, Jauniaux E, Campbell S, Pandya P, Cardy DL, Nicolaides KH: Cocloccntesis: a New Tcchniquc for Early Prenatal Diagnosis. Lancer 1993, 341:1623-1624.

54. Bianchi DW, Mahr A, Zickwolf GK, Houseal TW, Flint AF, Klinger KW: Detection of Fetal Cells with 47,XY,+21 Karyotypc in Maternal Feriphcral Blood. Hum Genet 1992, 90:368-370.

55. Zheng Y-L, Caner NP, Price CM, Colman SM, Milton PJ, . . Hackett GA, Greaves MF, Ferguson-Smith MA: Prenatal Dia-

gnosis from Maternal Blood; Simultaneous Immunophcno typing and FISH of Fetal Nucleated Etythrocytcs Isolated by Ncgativc Magnetic Cell Sorting. J Med G&wet 1993, 30:1051-1056.

‘382 Genetics of disease

This paper describes a pilot study into the usefulness of simultaneous immunophenotyping and FISH for the analysis of fet%I cells enriched from maternal circulation by magnetic activated ceII sorting (MACS). MACS is fast and inexpensive compared to flow sorting. In six preg- nandes, the predicted fetal sex agreed with conventional cytogenetic analysis of amniotic fluid cultures.

56. * Dclhanty JDA, Griffm DK, Handyside AH, Harper J, Atkin- son GHG, Pieters MHEC, Winston RML: Dctcction of Aneuploidy and ChromosomaI Mosaicism In Human Em bryos during PrcIIpIantation Sa Dctcrmhration by FIuo rcscent in S/tu Hybridization (FISH). Hum Md Genet 1993, 2:11831185. ,

57.

58.

59. .

Scherthan H, Loidl J, Schuster T. Schweizcr D: Mciotic Chro mosomc Condcnzation and Pairing in Succ&mmzyccs ~etrp vlslae Studied by Chromosome Painting. Cbromaromu 1992, 101:590-595. Goldman ASH, Huktn MA: Analysis of Chiasma Frcqucncy and First Mciotic Segregation in a Human Male Reciprocal Tramlocation Hctcroxygotc, t(l;ll> (p36.3;ql3.1), Using Flu- .orcsccncc in S&u Hybridization. Cytogenet Cell G&wet 1993, 63:16-23.

Ma C, MartIn S, Trask B, HamIin JL: Sister Chromatid Fusion Initiates Amplification of the Dihydrofolatc Rcductasc Gcnc in ChIncsc Hamster CcIIs. Genes Dar 1993, 7:60%20.

This paper examines early amplification events of the dihydrofolate reductasc gene in Chinese hamster cells using two-colour FISH with selected probes along the long arm of chromosome 2. The most frequent initiating event Is the formation of a giant Inverted duplication. The resultant dicentric chromosomes initiate breakage/fusion/bridge cycles that appear to mediate subsequent ampI&cation steps to higher copy number. 60. Lucas JN, Sachs RK: Using ThrccColor Chromosome Paint-

ing to Test Chromosome Aberration Models. J+oc Nat1 Acad SCf USA 1993, 90:1484-1487.

61. Vourc’h C, Tams&o D, Boyle AL, Ward DC: CcII Cyclc- Dcpcndcnt Distribution of Tclomcrcs, Ccntromcrcs and Chromosomc-Spccific SubsatcIIitc Domains in the Intcr- phase Nucleus of Mouse Lymphocytes. E*p Cell Res 1993, 205:142-151.

62. Caner KC, Bowman D, Carrington W, Fogarty K, McNeil JA, Fay FS, Lawrence JB: A ThrccDimcnsionaI View of Prc-

63.

64.

65.

66.

67.

68. . .

cursor Mcsscngcr RNA Metabolism within the Mammalian Nucleus. Sckmce 1993, 259:133&1335. Xing Y, Johnson CV, Dobner PR, Lawrence JB: Higher Lc- vcl Organization of IndIviduaI Gene Transcription and RNA Splicing. Science 1993, 259:1326-1330. Matera AG, Ward DC: Nuclcoplasmic Organization of SmaII Nuclear Ribonuclcoprotcins in Cuhurcd Human Cells. / Cell Btol 1993, 121:715-727. Selig S. Okumura K, Ward DC, Cedar H: Delineation of DNA Replication lime Zones by Fluorescence fn Situ Hy- bridization. EMBO / 1992, 11:1217-1225. KItsberg D, Selig S, Brandeis M, Simon 1, Kcshet I, Driscoll DJ, Nicholls RD, Cedar H: AIIclc-Spccitic Replication Tiig of Imprinted Gcnc Regions. Nature 1993, 364:459-463. Call LM, Migeon BR: DNA Replication AnaIysis of X-Linked FMR-1. XIST and Factor EC Loci Using Fluorcsccncc In Sftu Hybridization Indicates Non-Transcribed Gcncs Arc Late Replicating. Am J Hum Genet 1993, 53:224. Kitsbcrg D, Sclig S, Keshet I, Cedar H: Replication Struc- turc of the Human wlobin Gcnc Domain. Nature 1993, 366:588-590.

The authors present definitive evidence for a specific origin of rcpli- cation upstream of the g globin gene by analysis of hybridization patterns in Interphase nuclei. The origin is bidirectional and is used to initiate DNA synthesis when the domain replicates early in expressing cells and also when it replicates late in non-expressing cells. 69. Ried T, Rudy B, Vega Saeru de Miera E, Iau D, Ward DC,

Sen L: Localization of a Highly Conserved Human Potas- sium Channel Gcnc (NGK2NV4; KCMCl) to Chromosome 11~15. Cenomfcs 1993, 153405411.

70. KaIhoniemi OP, KaIIioniemi A, Sudar D, Rutovitz D, Gray JW, Waldman F, Pinkel D: Comparative Gcnomic Hybridi tion: a Rapid New Method for Dctccting and Mapping DNA Amplification in Tumors. Cancer Biol 1993, 4:41-46.

VJ Buckle and L Keamey. MRC Molecular Haematology Unit, Institute of Molecular Medicine, John Radcliffe Hospital, Hcadington, Oxford, OX3 9DU, UK.

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New methods in cytogenetics