This page has been archived and is no longer updated
Human cytogenetics: 46 chromosomes, 46 years and counting
Author: Barbara Trask
Keywords
Keywords for this Article
Add keywords to your Content
Save
|
Cancel
Share
|
Cancel
Revoke
|
Cancel
Rate & Certify
Rate Me...
Rate Me
!
Comment
Save
|
Cancel
Flag Inappropriate
The Content is
Objectionable
Explicit
Offensive
Inaccurate
Comment
Flag Content
|
Cancel
Delete Content
Reason
Delete
|
Cancel
Close
Full Screen
"� 2002 Nature Publishing Group ?Before a renewed, careful control has been made of the chromosome number in spermatogonial mitoses of man we do not wish to generalize our present findings into a statement that the chromosome number of man is 2n = 46, but it is hard to avoid the conclusion that this would be the most natural explanation of our observations.? 1 The field of human cytogenetics was launched in 1956 with this hesitant statement. The serendipitous addi- tion of water to a suspension of human mitotic cells 2 , before they were fixed and dropped onto glass micro- scope slides, caused the chromosomes to spread apart from each other so that Tjio and Levan 1 could accu- rately count the full complement of 46 human chro- mosomes (FIG. 1). The number 46 was independently confirmed by Ford and Hamerton in the same year 3 . The prevailing dogma had held the count at 48 for more than 30 years, ever since the geneticist Thomas Painter had reported on his observations of testicular cells 4 . Establishing the correct number and this simple technological advance set off many discoveries that associated specific chromosomal abnormalities with disease in the late 1950s and quickly established the central role of cytogenetics in medicine. In the ensuing years, human cytogenetics has been transformed by technological advances that have combined innovations in molecular biology, chem- istry and instrumentation. Cytogeneticists can now extract far more information about the human genome than just chromosome number. Each chro- mosome can be easily recognized ? even in the highly rearranged karyotypes of tumour cells ? by colour-coded labels. The resolution and sensitivity of analyses have improved more than 10,000-fold in a very short time, first with the introduction of banding technology and later with fluorescence in situ hybridization (FISH). Extremely subtle alterations in chromosome composition can now be detected and analysed for their association with disease. Cytogeneticists have been freed from their early dependence on mitotic cells by techniques that make it possible to evaluate the karyotype of non-dividing cells. Other approaches yield quantitative information on chromosomal content and structure and allow cytogeneticists to isolate specific chromosomes for molecular analyses. The latest technology allows genome-wide screens for the loss or gain of chromo- somal material to be conducted at unprecedented res- olution. Most importantly, the cytogenetic map is cross-referenced to the human draft sequence at thou- sands of points. These connections greatly facilitate the translation of microscopically visible clues of the molecular basis of disease to the actual genes that are disrupted or altered in dosage. HUMAN CYTOGENETICS: 46 CHROMOSOMES, 46 YEARS AND COUNTING Barbara J. Trask Human cytogenetics was born in 1956 with the fundamental, but empowering, discovery that normal human cells contain 46 chromosomes. Since then, this field and our understanding of the link between chromosomal defects and disease have grown in spurts that have been fuelled by advances in cytogenetic technology. As a mature enterprise, cytogenetics now informs human genomics, disease and cancer genetics, chromosome evolution and the relationship of nuclear structure to function. NATURE REVIEWS | GENETICS VOLUME 3 | OCTOBER 2002 | 769 Division of Human Biology, Fred Hutchinson Cancer Research Center, Seattle, Washington 98109, USA. e-mail: btrask@fhcrc.org doi:10.1038/nrg905 HUMAN GENETICS AND DISEASE REVIEWS � 2002 Nature Publishing Group KNUDSON?S TWO-HIT MODEL First proposed by Alfred Knudson in 1971, this model indicates that successive hits, such as deletion or mutation, in both alleles of a tumour- suppressor gene are required to turn a normal cell into a cancer cell. DUFFY BLOOD GROUP An antigenic variant of a chemokine receptor that is expressed on red blood cells. 770 | OCTOBER 2002 | VOLUME 3 www.nature.com/reviews/genetics REVIEWS 1959, trisomy 21 was shown to be the cause of Down syndrome 8 , and abnormalities in the number of sex chromosomes were shown to cause Turner syndrome (X0) and Klinefelter syndrome (XXY), two frequent dis- orders of sex differentiation 9,10 . It also became quickly apparent that most miscarriages were caused by abnor- malities in chromosome number 11 . Work on the diminutive, but deadly,?Philadelphia? chromosome established a new model for using cyto- genetic clues to find genes that, when altered, cause human disease. In 1960, cytogeneticists recognized the Philadelphia chromosome as the cause of chronic myeloid leukaemia (CML) 12 . Thirteen years later, this chromosome was shown by Janet Rowley to be the product of a translocation between chromosomes 9 and 22 (REF. 13). The point at which these two chromo- somes break and fuse was the obvious place to look for the molecular explanation of this disease. Indeed, by using the derivative chromosomes in molecular assays, the translocation was shown, in 1985, to create a new hybrid gene of BCR and ABL (breakpoint cluster region and v-abl Abelson murine leukaemia viral oncogene homologue 1) 14 . Subsequent studies showed that constitutive activation of BCR?ABL, a tyrosine kinase, affects many cellular pathways and leads to the cancer phenotype (reviewed in REF. 15). This under- standing in turn led to the development of Gleevec (STI571), a drug that was designed to block the func- tion of the BCR?ABL protein and that has proved to be a highly successful treatment for CML 16 . The rudimentary chromosome preparations of the early 1960s yielded other breakthroughs in human genetics. Lejeune recognized the first inher- ited deletion syndrome, Cri du Chat, in 1963; patients with severe mental retardation and a characteristic cat-like cry were all missing a portion of the short arm of chromosome 5 (REF. 17). In the same year, a patient with bilateral retinoblastoma was found to have a deletion of the long arm of a D-group chro- mosome 18 . Later work by Cavenee et al. 19 provided paradigm-setting proof of KNUDSON?S TWO-HIT HYPOTHESIS 20 by showing that the cancer arises owing to the loss of one allele of the RB (retinoblastoma) gene in 13q14 and mutation of the other allele. One of the first autosomal human genes to be mapped, the gene for the DUFFY BLOOD GROUP, was assigned to chromosome 1 because of the consistent way it tracked in families as a visible cytogenetic anomaly near the centromere of chromosome 1 (REF. 21). Chromosomal barcodes The power of cytogenetic analysis redoubled in the late 1960s with Torbjorn Caspersson?s development of staining protocols that produced highly reproducible patterns of dark and light bands along the length of each chromosome 22 . These banding patterns became the barcodes with which cytogeneticists could easily identify chromosomes, detect subtle deletions, inver- sions, insertions, translocations, fragile sites and other more complex rearrangements, and refine break- points (FIG. 2). This article outlines the history of the main techno- logical advances that have occurred in human cytoge- netics during the past 46 years. It highlights the impact that these advances have on our understanding of the molecular basis of human disease and of the structure, function and evolution of our chromosomes. A late start, but rapid recovery Flemming and Arnold first observed human chromo- somes in the 1880s. It is therefore remarkable that such a fundamental aspect of human biology as chro- mosome number could have escaped the scientific community until 1956, three years after the structure of the DNA helix was elucidated 5 . Friedrich Vogel and Arno Motulsky 6 ascribe this delay to both technologi- cal and politico-social causes. They assert that most laboratory-based medical scientists at the time were uninterested in human genetics; they considered humans to be far too complex and preferred to focus on simpler model organisms that could be more easily manipulated. Also, many serious geneticists had disso- ciated themselves from human genetics during the eugenics movement in the early 1900s, which reached its nadir with the horrific practices of the Nazis. However, soon after the number 46 was firmly estab- lished, scientists readily applied the new cytogenetic technique to the investigation of phenotype?genotype correlations in humans and began to tap useful infor- mation from naturally occurring chromosomal rearrangements. Human cytogeneticists were dealt a good hand by evolution. Had human chromosomes been as morpho- logically similar as those of mice, or as tiny and numer- ous as those of most birds, progress in cytogenetics would have been much slower. Fortunately, differences in the relative size of human chromosomes and the position of the centromeric constriction allowed cyto- geneticists to match up the 23 pairs and classify them into seven groups (A to G) with relative ease 7 . Although crude, these early karyotypes allowed the discovery that some human disorders result from changes in chromosome number or appearance. In Figure 1 | The picture that established 46 as the chromosome number in man. Reproduced with permission from REF. 1 � (1956) Mendelian Society of Lund for the Scandinavian Association of Genetics. � 2002 Nature Publishing Group NATURE REVIEWS | GENETICS VOLUME 3 | OCTOBER 2002 | 771 REVIEWS Through the painstaking analysis of chromosome banding patterns, thousands of chromosomal abnor- malities have been associated with inherited or de novo disorders, generating many leads to the underlying molecular causes of these disorders (see Online links box at the end of the article). Even today, when high- resolution genetic linkage analysis can be conducted easily, the discovery of a patient whose disorder is caused by a gross chromosomal abnormality is her- alded as a valuable resource for locating the disease gene. Solid tumours also present a myriad of complex chromosomal aberrations ? each is a possible clue to tumour initiation and progression. The challenge is to navigate from the visible morphological alteration to the sequence level. The next major advances in cytoge- netics facilitated that process. Moving from microscope to molecule Once a rearranged chromosome has been identified, the next step is to position the translocation breakpoints or deletion boundaries relative to genes on molecular maps. This step can be accomplished by using tech- niques that physically separate abnormal and normal chromosomes so that they can be independently assayed for gene content. Three methods have been par- ticularly useful in achieving this: somatic-cell-hybrid technology, fluorescence-activated cell (chromosome) sorting (FACS) and FISH (all discussed below). These techniques help researchers to zoom in on the defect from the cytogenetic to the molecular level, and, impor- tantly, they have yielded rough maps for navigating the genome and for allowing more detailed molecular map- ping and sequencing. Somatic-cell hybrids are a fortunate quirk of cell biology. When rodent and human cells are fused in the laboratory, human chromosomes are preferentially ejected, but some are retained 25,26 . This phenomenon was capitalized on by the groups of Weiss and Ruddle, who were the first to use panels of hybrid cell lines, each retaining a different set of human chromosomes, to map genes and anonymous markers to specific chromo- somes or portions thereof 27,28 . The chromosomal con- tent of each line, established by cytogenetic analysis, is simply correlated with the results of hybridization assays, functional tests or PCR to assign a gene or marker of interest to a chromosome. Much more pre- cise maps, which served as frameworks for the assembly of the human genome sequence, were generated using panels that contain different chromosomal fragments, such as aberrant chromosomes transferred from the cells of patients 29 or fragments that were experimentally produced by radiation 30 . Originally developed for cell analysis and separa- tion, flow cytometry was adapted in 1979 for the quantitative analysis and sorting of human chromo- somes by a team of investigators at the Lawrence Livermore National Laboratory in California 31 . In this technique, chromosomes are released into suspension from mitotic cells and stained with two fluorescent DNA dyes that have different base-pair specificities: this allows all but four human chromosomes (9?12) The bands appear only in metaphase chromosomes, and cycling cells are therefore required for this analysis. If cells can be caught in prometaphase ? when chromo- somes are in the very early stages of condensation ? up to 2,000 bands can be discerned 23 ; more typically, 400?800 bands are visible. The band-naming convention introduced in 1971 reflects the levels of resolution with which chromosomes can be analysed 24 . Despite the extensive use of these bands, their cause remains an enigma. They correlate with regional differences in base- pair composition, repetitive elements, replication timing and chromatin packaging and can be induced by many methods, but their molecular basis is not understood. Cytogenetic information moved from the bench to the clinic in the late 1960s with the discovery that fetal cells could be obtained through AMNIOCENTESIS and could be checked for chromosomal abnormalities. Methods were quickly developed to induce fetal cells that had been derived from amniotic fluid to divide in culture and to obtain high-quality banded karyotypes. The same procedures are widely used today to provide pre- natal diagnostic information to families. AMNIOCENTESIS A procedure in which a small sample of amniotic fluid is drawn out of the uterus through a needle inserted into the abdomen. The fluid is then analysed to detect genetic abnormalities in the fetus or to determine the sex of the fetus. G-BANDS/R-BANDS Chromosome banding pattern produced by Giemsa staining (G-bands); the reciprocal pattern (reverse or R-bands) can be produced with various other staining procedures. 711 a b Figure 2 | Cytogenetic banding patterns of human chromosomes. a | An R-BANDED metaphase spread. b | G-BANDED chromosomes 7 and 11 from an individual with acute myeloid leukaemia, showing the subtle translocation that involves the terminal bands of the p (short) arms ? t(7;11)(p15;p15). This translocation generates a hybrid gene of NUP98 (nucleoporin 98 kDa) and HOXA9 (homeobox gene A9), which results in leukaemogenesis. Panel a was provided by Cynthia Friedman, Fred Hutchinson Cancer Research Center; panel b was provided by Diane Norback and colleagues at the Waisman Cytogenetics Center at the University of Wisconsin. � 2002 Nature Publishing Group 772 | OCTOBER 2002 | VOLUME 3 www.nature.com/reviews/genetics REVIEWS The FISHing trip The next advance to revolutionize cytogenetics, FISH, provided a direct link between microscope and sequence. This technique allows the chromosomal and nuclear locations of specific DNA sequences to be seen through the microscope (FIG. 4). Each probe is a cloned piece of the genome that is conjugated to a reporter molecule, such as biotin. After denaturation, the probe is allowed to seek out its complement in the chromo- somal DNA, and these locations are then marked with a fluorescent reagent, such as avidin-FITC, that binds to the reporter attached to the DNA probe. Although radioactively labelled DNA and RNA probes had been localized to cytogenetic bands since 1969 (REFS 40,41), the field advanced significantly when groups led by David Ward and Mels van der Ploeg replaced the isotopic labels with fluorescent ones 42,43 . Fluorescent tags are to be resolved by a flow cytometer 32 (FIG. 3). The mea- surements give quantitative information on the extent of normal variation in chromosome size (some vary by 50% in DNA content) and the amount of DNA that is missing or gained in abnormal chromo- somes 33,34 . Abnormal and normal chromosomes can also be separated for the molecular characterization of DNA-marker retention or loss 35 . Flow sorting was the key to the production of chromosome-specific DNA clone libraries 36,37 , which have been important for constructing detailed, marker-dense physical maps of the genome, especially in the days when tackling the whole genome at once seemed too daunting. Flow sorting continues to be the technique of choice for producing chromosome-specific paints 38 (see below) and for characterizing sequences that are duplicated on more than one chromosome 39 . ? + Deflection plates Collection vessel Waste- collection vessel 458-nm laser Sheath fluid Piezoelectric transducer Photomultiplier Pulse-height analysis, sorting circuitry Sample UV laser Lens Sort region 3 1 2 4 5 6 7 8 9-12 13 14 15 16 17 19 18 20 21 22 X Hoechst 33258 fluorescence intensity Chromomycin A3 fluorescence intensity Figure 3 | Schematic of the discrimination and sorting of human chromosomes by flow cytometry. Chromosomes that are released from mitotic cells are stained with two DNA-binding dyes with different base-pair specificities, and the fluorescence intensities of each of several thousand chromosomes are measured in a two-laser flow cytometer. In the example shown, the two dyes are Hoechst 33258, which binds preferentially to A?T base pairs, and chromomycin A3, which binds to C?G base pairs. The resulting bivariate ?flow karyotype? (bottom right panel) resolves all chromosomes except for the 9?12 group. In this example, maternal and paternal homologues of both chromosomes 21 and 19 are resolved into separate peaks owing to differences in their DNA content. After measurement, droplets that contain desired chromosomes, such as chromosome 3 in this example (white box), can be deflected into tubes for molecular analyses. UV, ultraviolet. Diagram modified with permission from REF. 95 � (1986) Cold Spring Harbor Laboratory Press; plot provided by Ger van den Engh, Institute of Systems Biology. � 2002 Nature Publishing Group NATURE REVIEWS | GENETICS VOLUME 3 | OCTOBER 2002 | 773 REVIEWS The interplay between genome mapping and cyto- genetics escalated in the mid-1980s as FISH technol- ogy improved and cloned DNA reagents became avail- able through the efforts of many genome mapping and sequencing groups. Using FISH, cytogeneticists could detect chromosomal abnormalities that involve small segments of DNA ? if their probe was situated, fortu- itously or by design, in the affected chromosomal seg- ment 51 . Cytogeneticists were no longer limited to the resolution afforded by crude banding patterns. Conversely, FISH could be used to establish the order of DNA clones relative to bands, naturally occurring breakpoints and other clones (for example, REF. 52) (FIG. 4). These data were funnelled into the genome project as independent tests of the validity of maps constructed by other techniques 53 . In turn, as molecu- lar biologists filled in the genome maps, large collec- tions of molecular reagents in the form of cloned, mapped segments of the human genome (cosmids, BACS, PACS AND YACS) became available with which abnor- mal chromosomes could be characterized by FISH to identify affected genes. For example, FISH analyses identified clones that cross the two breakpoints of the PERICENTRIC INVERSION of chromosome 16 seen in patients with acute myelogenous leukaemia (AML). This find- ing set the stage for the identification of the two genes (MYH11, smooth muscle myosin heavy chain 11, and CBFB, the ?-subunit of core-binding factor) that, when aberrantly fused, cause the leukaemic transfor- mation 54,55 . Cytogenetic studies in Sam Latt?s labora- tory were crucial to the discovery that Angelman and Prader?Willi syndromes are disorders of IMPRINTING: rearrangements in 15q11?15q13 were invariably found in the maternal or paternal copy of this region, respec- tively 56 , and FISH has been crucial in the identification of imprinted genes in this region (reviewed in REF. 57). The genome-wide view afforded by FISH has also revealed sequences that have been duplicated at distinct sites in the human genome; these sequences light up at more than the two expected sites and can be flagged for special attention during the assembly of the draft sequence 53 . Furthermore, many of these duplicated blocks have been implicated in chromosomal rearrange- ments that cause disease and are therefore of biological interest (reviewed in REF. 58). Even more importantly, FISH opened up the nuclei of non-dividing cells to karyotype analysis. Conventional cytogenetics requires the capture of cells in mitosis, and many samples, particularly those from solid tumours, produce few, if any, analysable metaphases. Using FISH and chromosome-specific probes, cytogeneticists can enumerate chromosomes, simply by counting spots in each nucleus 59,60 . Deviations in spot number also signal gene deletion and amplification. Because DNA is pack- aged ~10,000-fold more loosely in interphase nuclei than in metaphase chromosomes, abnormalities that are not resolvable by metaphase FISH, such as the 1-Mb duplication that causes CHARCOT?MARIE?TOOTH SYNDROME 61 , can be detected by interphase FISH (FIG. 5). Shifts in rela- tive spot position reveal structural rearrangements, such as translocations and inversions 62 (FIG. 5). safer and simpler to use, can be stored indefinitely, give higher resolution and opened up prospects for simulta- neously locating several DNA sequences in the same cell by labelling them with different fluorochromes. In less than 15 years, the sensitivity of FISH improved 10,000-fold. This remarkable achievement can be attributed to improvements in the probe labels that made them less bulky, simpler to incorporate into the probe and brighter; in the optics for fluorescence microscopy; and in more mundane, but crucial, aspects of the procedure, such as probe fragmentation and slide storage. By 1985, the first single-copy human gene, thy- roglobulin, had been localized to a chromosome band by non-radioactive in situ hybridization 44 . This feat was an important milestone, even though thyroglobulin was one of the largest genes known at the time, and speci- ficity was achieved by fastidiously removing all the interspersed repetitive elements from the probe before its use. Today, localizing segments as small as 10 kb is routine and 1 kb is achievable 45 . We now exploit the kinetics of DNA reassociation to pre-anneal the repeti- tive elements, so that only the unique/low-copy por- tions of the labelled probe are available for hybridization to chromosomes 41,46 . Using new probes that are based on PEPTIDE NUCLEIC ACID chemistry, the intensity of FISH spots is a reasonable measure of the local amount of complementary target. A good illustration is the study of telomere dynamics in normal and immortalized cells by quantitative analyses of TTAGGG-specific probes bound to the ends of chromosomes 47 . A clever modifi- cation of FISH (called COD-FISH) goes even further to reveal the absolute 3??5? direction of a particular sequence on the chromosome 48 and to detect inversions and sister-chromatid exchanges 49,50 . PEPTIDE NUCLEIC ACID (PNA). An analogue of DNA in which the backbone is a pseudopeptide rather than a sugar. PNA mimics the behaviour of DNA, but, because PNA has a neutral backbone, it binds complementary nucleic- acid strands more strongly and with greater specificity than an oligonucleotide. COD-FISH (Chromosome orientation and direction-fluorescence in situ hybridization). In this technique, single-stranded probes hybridize to one chromatid of a metaphase chromosome, because the most recently synthesized strand in each chromatid is specifically degraded before hybridization. A probe that recognizes the cytosine-rich strand of the telomeric repeat provides orientation by marking the 5?-end of each chromatid. BAC, PAC AND YAC Cloning vector system able to accomodate large genomic fragments. BACs and PACs are grown in bacteria; YACs are grown in yeast. PERICENTRIC INVERSION A structural alteration to a chromosome that results from breakage, inversion and reinsertion of a fragment that spans the centromere. ab Figure 4 | Cytogenetic localization of DNA sequences with fluorescence in situ hybridization (FISH). a | FISH produces a fluorescent signal (red) at the sites of a specific DNA sequence; in this case, a 150-kb segment of chromosome 1. Reproduced with permission from Nature REF. 53 � (2001) Macmillan Magazines Ltd. b | Several probes, each corresponding to a defined genomic segment, can be simultaneously analysed and ordered with respect to each other using multicolour FISH. Reproduced with permission from REF. 96 � (2002) Springer Verlag. Provided by Ullrich Weier, Lawrence Berkeley National Laboratory. � 2002 Nature Publishing Group 774 | OCTOBER 2002 | VOLUME 3 www.nature.com/reviews/genetics REVIEWS Clinical cytogenetics laboratories now make signifi- cant use of FISH in both their diagnostic and their research work. FISH is routinely used to augment con- ventional banding analyses of chromosomal rearrange- ments. Cytogeneticists have at their disposal various commercially available probe kits that are tailored for specific questions, such as the diagnosis of syndromes caused by chromosomal abnormalities that are too sub- tle to detect reliably by banding. The FISH test for SMITH?MAGENIS SYNDROME, which uses a probe for a small deleted region of chromosome 17, is an excellent exam- ple 68 . In research, FISH features prominently in the cyto- geneticists? process of finding recurrent translocation breakpoints or overlapping deletions among patients with similar phenotypic abnormalities. Chromosome painting with a colourful palette The thrill of seeing a single-copy gene fluoresce in a human cell was soon surpassed by the vivid image of 24 human chromosomes painted in different colours 69,70 (FIG. 6). This powerful development, called spectral kary- otyping (SKY) or multiplex (M)-FISH, combines three significant advances. First was the production of chromo- some-specific ?paints?: collections of sequences derived from each chromosome (usually by flow sorting) 71,72 . These collections can be generated easily from small numbers of chromosomes using DEGENERATE OLIGONUCLEOTIDE-PRIMED PCR 38 or LINKER?ADAPTOR PCR 73 .When used as a probe, these collections label a chromosome end to end. (Region-specific paints can be generated if microdissected portions of chromosomes are used as a template 74 .) Second was the combination of fluo- rochromes to produce 24 colour combinations, one for each chromosome 75 . Third were the advances in micro- scopic optics, filters and imaging systems for multicolour analyses. In the SKY system, the spectral characteristics of each pixel in the image are read out by an INTERFEROMETER 69 . In M-FISH, the spectral characteristics are evaluated by collecting images through a series of excitation and emis- sion filters 70 . These imaging systems can be taught to clas- sify each chromosomal segment automatically, and they offer the first real hope of automated karyotype analysis. So far, no system can classify banded chromosomes as robustly and accurately as a skilled cytogeneticist, despite the millions of dollars that have been invested in auto- mated karyotype analysis since 1968. SKY and M-FISH have proved to be extremely useful for detecting translocations and other complex aberra- tions (FIG. 6). For example, SKY has revealed amplifica- tion of regions on 11q, 21q and 22q that had not been detected before in AML patients with complex kary- otypes; these defects could have a significant role in leukaemogenesis 76 . Even the karyotypes of tumours in mice can be deciphered 77 . M-FISH has been especially helpful in the study of radiation-induced damage and chromosome repair 78 . Although the breaks occur ran- domly, they are repaired in non-random patterns that reflect the proximity of the breaks in the nucleus during the repair process. So, SKY both has an impact on radia- tion dosimetry and gives insights into the organization of the human cell nucleus 79 . Interphase FISH has also made it possible to deter- mine the relative times at which specific DNA sequences are replicated during the S phase of the cell cycle. Before replication, the probe generates a single dot on each chromosome, whereas two closely juxtaposed dots are visible after replication 63 . Using this approach, it was found that the order of replication is carefully orches- trated, and, for most loci, that the maternal and paternal alleles replicate in synchrony. By contrast, alleles of most imprinted loci are asynchronously replicated, with the expressed allele replicating earlier than the silenced one 64 . As the relationship between sequence proximity in interphase chromatin and separation along the DNA helix was elucidated, the order of DNA sequences could be inferred with 50?100-kb resolution by measuring the distances between fluorescent spots that mark DNA sequences of interest 65 . The ultimate in cytogenetic reso- lution is reached by wiping out nuclear organization altogether and conducting FISH on DNA fibres that have been affixed to glass (fibre-FISH) 66,67 . What is con- densed to a small spot at the resolution of light microscopy in interphase becomes a long fluorescent line in fibre-FISH. Fibre-FISH is used to resolve ambi- guities in the order of genes in a chromosomal region, to analyse the organization of tandem duplications and to detect small-scale rearrangements in chromosomes. IMPRINTING A genetic mechanism by which genes are selectively expressed from the maternal or paternal homologue of a chromosome. CHARCOT?MARIE?TOOTH SYNDROME An inherited degenerative peripheral nerve disorder that causes progressive muscle weakness and atrophy in the feet, legs, hands and forearms. SMITH?MAGENIS SYNDROME A rare condition that is associated with developmental delay, characteristic facial and other anatomical abnormalities, learning difficulties and behavioural problems, such as the tendency to harm oneself. ab 917 der(22) der(9) 22der(17) Figure 5 | Using FISH to detect chromosomal abnormalities in interphase nuclei. a | The duplication of a small portion of chromosome 17 that causes Charcot?Marie?Tooth syndrome is evident from the appearance of three, rather than two, red signals in this nucleus. The green spots mark a sequence outside the duplication. b | The translocation that creates a fusion of the BCR (breakpoint cluster region; on chromosome 22) and ABL (v-abl Abelson murine leukaemia viral oncogene homologue; on chromosome 9) genes in Philadelphia-chromosome-positive chronic myeloid leukaemia is evident from the close juxtaposition of one pair of green and red signals. These signals were generated using FISH (fluorescence in situ hybridization) probes for sequences located near these two genes, respectively. der(22) is the Philadelphia chromosome. Only the relevant portions of the normal and abnormal chromosomes are shown in the diagram below each panel. der, derivative. The photo in a is modified from REF. 61 � (1991) Elsevier Science; the photo in b is reproduced from REF. 62 � (1990) American Association for the Advancement of Science. � 2002 Nature Publishing Group NATURE REVIEWS | GENETICS VOLUME 3 | OCTOBER 2002 | 775 REVIEWS disease-gene mapping. The use of dogs to identify genes that cause human disease is a case in point 85 .At least half of the inherited disorders that are recognized in various dog breeds correspond to specific human diseases, including various forms of cancer, deafness, heart disease, blindness and epilepsy. With extensive dog pedigrees, it is feasible to genetically map the canine disease to a region of the dog genome. Comparative cytogenetic maps of the human and dog genomes, produced by hybridizing human chromo- some paints to dog chromosomes 86 , show where to dig in the human genome for candidate genes, which can then be tested for mutations in dogs and/or humans. CGH-arrays ? a surrogate for chromosomes The next transformation of cytogenetics came with the realization that genome-wide scans for the loss or gain of chromosomal material could be conducted without even looking directly at the subject?s chromosomes. The technique that made this possible is called com- parative genome hybridization (CGH) and was devel- oped by a team led by Ollie and Anna Kallioniemi, Dan Pinkel and Joe Gray 87 . In this approach, the genomic DNA of test and reference samples is isolated, fragmented, labelled in red and green, respectively, and allowed to compete for hybridization sites in sets of normal chromosomes (FIG. 7). As in regular FISH, interspersed repetitive elements are taken out of the picture by pre-annealing the probes with unlabelled DNA that is enriched for repetitive sequences. The ratio of red-to-green fluorescence is measured along the length of each chromosome. The chromosomal regions that are equally represented in the test and ref- erence samples appear orange, but those deleted or amplified in the test sample appear more red or more green. CGH is particularly important in cancer cyto- genetics, in which it is used to identify chromosomal regions that are recurrently lost or gained in tumours. For example, CGH led the way to the identification of PIK3CA, the catalytic subunit of phosphatidylinositol 3-kinase (PI3K), as an oncogene in ovarian cancer 88 . DNA-amplification techniques have also been devel- oped to find genetic alterations in small samples of rare cells 89 , such as rogue cells found in blood that have escaped a primary tumour and might foreshadow metastasis. The current excitement in cytogenetics revolves around the promise of array-CGH 90 (FIG. 7). In this technique, metaphase chromosomes are replaced by an array of thousands of BAC clones, each of which con- tains an ~150-kb segment of the human genome. An array of 3,000 BACs can be constructed that samples the genome, on average, once every megabase pair 53 . Array-CGH is therefore the equivalent of conducting thousands of FISH experiments at once, but without the need to count dots to measure the copy number of each test locus. CGH provides better quantification of copy number and more precise information on the breakpoints of segments that are lost or gained than does conventional CGH. More importantly, each clone is an entry point to the genomic sequence in which M-FISH has also sparked a new industry of probe development to monitor many loci at once for subtle aberrations. The best example is the use of probes that mark the unique sequence near each telomere to detect subtle rearrangements of the ends of chromosomes 80 . With this technique, as many as 7% of patients with pre- viously unexplained mental retardation have been found to have chromosome abnormalities that had gone undetected in previous analyses 81 . One of the most thriving areas of cytogenetics today is the study of the chromosomal rearrangements that occurred during evolution 82,83 . During each speci- ation event, some cards in the genome deck are moved. These events can be reconstructed with FISH. Such studies have revealed, for example, that the evolu- tionary rate of chromosomal translocations is ten times greater between the mouse and the rat genomes than between those of humans and cats or chim- panzees 84 . Comparative cytogenetics is also crucial for DEGENERATE OLIGONUCLEOTIDE-PRIMED (DOP) PCR/LINKER?ADAPTOR PCR DOP-PCR uses partially degenerate primers to amplify sequences at dispersed sites in a sample. In linker-adaptor PCR, the DNA sample is digested with a restriction enzyme, the ends are ligated to an adaptor oligonucleotide, and the ligated fragments are amplified using PCR primers that are complementary to the linker- adaptor oligonucleotide. Both techniques generate large pools of fragments that almost completely represent the starting sample. 1 2 3 22 X Y + Cot-1 DNA Labelling of the individual chromosome- painting probes using various combinations of fluorochromes 24 flow-sorted human chromosomes Probe mixture, which contains all of the differentially labelled chromosome- painting probes Hybridize, detect and analyse using interferometer or multiple fluorescence filters Metaphase chromosome preparation a b c Figure 6 | Spectral karyotyping and multicolour-FISH paint each human chromosome in one of 24 colours. a | Outline of the spectral karyotyping (SKY) protocol. SKY and multicolour fluorescence in situ hybridization (M-FISH) differ only in the method used to measure the spectral characteristics of each pixel in the image (see main text). Cot-1 DNA is enriched in repetitive sequences, and by binding to repetitive sequences in the fluorescently tagged probes, it suppresses their hybridization to target chromosomes. b | The application of SKY to normal interphase and metaphase human cells; the highly rearranged karyotype of a bladder cancer cell is shown in c. Arrows point to inter-chromosomal rearrangements. Panels a and b modified from REF. 97 � (2000) Cambridge University Press. Panel c reproduced from REF. 98 � (1999) Wiley. � 2002 Nature Publishing Group 776 | OCTOBER 2002 | VOLUME 3 www.nature.com/reviews/genetics REVIEWS markers, identify new tumour-suppressor genes or oncogenes and, ultimately, lead to a better understand- ing of the cancer process. In addition, I predict that some prenatal diagnostic tests that now rely on banding and conventional FISH will also be supplanted by cus- tom arrays. It is hoped that technological advances, affected genes can be identified. Although CGH is insensitive to changes that are present at low frequency in the cells being analysed, it is expected that array- CGH will enable many groups to evaluate large num- bers of tumours for recurrent changes using a common platform. These analyses should generate prognostic INTERFEROMETER A device that uses an interference pattern to determine wave frequency, length or velocity. Ratio green:red 1p 1p XqPosition in genome 1 2 3 6q 11q X 20p 20q12 13 Tumour cells Extract, amplify and label DNA Hybridize to metaphase spread or BAC array 1pter Xqter Normal cells 1 7 18 13 8 2 14 19 9 3 15 20 10 4 16 21 11 5 17 22 6 12 X Figure 7 | Comparative genome hybridization. This technique reveals the loss or gain of chromosomal regions in test samples (for example, derived from a tumour) relative to normal controls. DNA in the test and reference samples is labelled with green and red fluorochromes, respectively, and allowed to compete for hybridization sites on either metaphase chromosomes (left) or an array of BAC (bacterial artificial chromosome) clones that represent thousands of small DNA segments distributed across the genome (right). Areas on the chromosome, or spots on the array, that are more green than average are present in extra copies in the test sample; those that are more red than average are deleted in the test sample. The bottom left panel shows the outcome of conventional comparative genome hybridization using the prostate-cancer cell line PC-3 as a test sample; the loss and gain of several chromosomal regions are evident as red and green areas, respectively. Inset: the profile of the average green:red ratio of ten chromosomes shows 8p loss and 8q gain in these cells. The bottom right panel shows copy-number losses of 1p, 6q, 11q and 20p, and gains of 12, 13 and 20q in the ML-2 cancer cell line. Bottom left panel provided by Ilona Holcomb, Fred Hutchinson Cancer Research Center; bottom right panel reproduced with permission from Nature REF. 53 � (2001) Macmillan Magazines Ltd. � 2002 Nature Publishing Group NATURE REVIEWS | GENETICS VOLUME 3 | OCTOBER 2002 | 777 REVIEWS tumour biology, by opening up the possibility of assess- ing aneuploidy, as measured by the fluorescence of chromosome-specific probes, in conjunction with other phenotypic characteristics of cells, such as cell- surface antigens. Finally, techniques to mark endoge- nous loci in live cells and new applications of genome- wide array technology (such as in REFS 93,94) are needed to investigate how chromosome and nuclear structure relates to gene regulation, replication and repair. Recurrent reincarnation Human cytogenetics is flourishing at the age of 46 years and does not risk being supplanted by younger and more precise molecular techniques. Chromosomal abnormalities are nature?s guide to the molecular basis of far too many unexplained human disorders, particu- larly solid tumours. Furthermore, cytogenetics contin- ues to reinvent itself to aid explorations of chromosome structure, function and evolution. The cytogenetics lab is a bustling enterprise of service and research, with a still expanding set of tools. Banding techniques, which are unchanged from the 1970s apart from the introduc- tion of digital image handling, are now combined with state-of-the-art multicolour FISH and molecular analy- sis. From their vantage through the microscope, the cytogeneticists? view of the genome is still unrivalled in its scope, detail and colour. such as array-CGH, will reduce the time and cost of cytogenetic analyses so that they can be accessed by more families. What next? Of course, cytogenetic technology has its limitations. Ideally, each cytogenetics lab should have at hand a bank of clones that represent sequences that are distrib- uted once every megabase-pair across the genome, so that any chromosomal abnormality could be analysed at the molecular level with ease and efficiency using conventional FISH or array-CGH. Efforts have been made to assemble and distribute such a reagent set 53 . Navigating from the microscope to the DNA sequence and back again would be further facilitated by increased FISH sensitivity, so that probes as short as most PCR products could be reliably detected. Signal- amplification techniques, such as those that involve TYRAMIDE CHEMISTRY 91 or rolling-circle amplification 92 ,are pushing at this limitation. The study of duplications by FISH would be more informative with a clearer under- standing of how variation in target size and divergence affects signal intensity. Strategies for robust allele- specific FISH might allow determination of the posi- tion and copy number of maternal or paternal alleles. The adaptation of FISH for flow cytometric analysis of cells would greatly benefit our understanding of TYRAMIDE CHEMISTRY A labelling system that uses a hybridization probe that is directly or indirectly labelled with peroxidase. The peroxidase catalyses the localized deposition of a reactive tyramide-labelled tag (for example, biotin or fluorescent dyes). 1. Tjio, H. J. & Levan, A. The chromosome numbers of man. Hereditas 42, 1?6 (1956). This paper provides the first correct count of human chromosome number, which was independently confirmed by Ford and Hamerton (reference 3) in the same year. 2. Hsu, T. C. Human and Mammalian Cytogenetics: an Historical Perspective (Springer, New York, 1979). 3. Ford, C. E. & Hamerton, J. L. The chromosomes of man. Nature 178, 1020?1023 (1956). 4. Painter, T. S. Studies in mammalian spermatogenesis. II. The spermatogenesis of man. J. Exp. Zool. 37, 291?321 (1923). 5. Watson, J. R. D. & Crick, F. H. C. A structure for deoxyribose nucleic acid. Nature 171, 737?738 (1953). 6. Vogel, F. & Motulsky, A. G. Human Genetics: Problems and Approaches (Springer, Berlin, 1997). 7. Chicago Conference 1966. Standardization in Human Cytogenetics. Birth Defects: Original Article Series Vol. 2, No. 2 (The National Foundation, New York, 1966). 8. Lejeune, J., Gautier, M. & Turpin, M. R. Etude des chromosomes somatiques de neuf enfants mongoliens. C. R. Acad. Sci. (Paris) 248, 1721?1722 (1959). 9. Ford, C. E., Miller, O. J., Polani, P. E., de Almeida, J. C. & Briggs, J. H. A sex-chromosome anomaly in a case of gonadal dysgenesis (Turner?s syndrome). Lancet 1, 711?713 (1959). 10. Jacobs, P. A. & Strong, J. A. A case of human intersexuality having a possible XXY sex-determininig mechanism. Nature 183, 302?303 (1959). 11. Clendenin, T. M. & Bernirschke, K. Chromosome studies on spontaneous abortions. Lab. Invest. 12, 1281?1292 (1963). 12. Nowell, P. C. & Hungerford, D. A. A minute chromosome in human chronic granulocytic leukemia. Science 132, 1497?1501 (1960). 13. Rowley, J. D. A new consistent chromosomal abnormality in chronic myelogenous leukaemia identified by quinacrine fluorescence and Giemsa staining. Nature 243, 290?293 (1973). 14. Heisterkamp, N., Stam, K., Groffen, J., de Klein, A. & Grosveld, G. Structural organization of the bcr gene and its role in the Ph? translocation. Nature 315, 758?761 (1985). 15. Deininger, M. W., Goldman, J. M. & Melo, J. V. The molecular biology of chronic myeloid leukemia. Blood 96, 3343?3356 (2000). 16. Druker, B. J. Perspectives on the development of a molecularly targeted agent. Cancer Cell 1, 31?36 (2002). 17. Lejeune, J. et al. Trois cas de deletion partielle du bras court d?un chromosome 5. C. R. Acad. Sci. (Paris) 257, 3098?3102 (1963). 18. Lele, K. P., Penrose, L. S. & Stallard, H. B. Chromosome deletion in a case of retinoblastoma. Ann. Hum. Genet. 27, 171?174 (1963). 19. Cavenee, W. K. et al. Expression of recessive alleles by chromosomal mechanisms in retinoblastoma. Nature 305, 779?784 (1983). 20. Knudson, A. G. Jr. Mutation and cancer: statistical study of retinoblastoma. Proc. Natl Acad. Sci. USA 68, 820?823 (1971). 21. Donahue, R. P., Bias, W. B., Renwick, J. H. & McKusick, V. A. Probable assignment of the Duffy blood group locus to chromosome 1 in man. Proc. Natl Acad. Sci. USA 61, 949?955 (1968). 22. Caspersson, T. et al. Chemical differentiation along metaphase chromosomes. Exp. Cell Res. 49, 219?222 (1968). This paper introduces the technique for identifying chromosomes by their banding pattern, a revolutionary step in human cytogenetics. 23. Yunis, J. J. Mid-prophase human chromosomes. The attainment of 2000 bands. Hum. Genet. 56, 293?298 (1981). 24. Paris Conference 1971. Standardization in Human Cytogenetics. Birth Defects: Original Article Series Vol. 8, No. 7 (The National Foundation, New York, 1972); also in Cytogenetics 11, 313?362 (1972). 25. Harris, H. & Watkins, J. F. Hybrid cells from mouse and man: artificial heterokaryons of mammalian cells from different species. Nature 205, 640?646 (1965). 26. Ephrussi, B. & Weiss, M. C. Interspecific hybridization of somatic cells. Proc. Natl Acad. Sci. USA 53, 1040?1042 (1965). 27. Weiss, M. C. & Green, H. Human?mouse hybrid cell lines containing partial complements of human chromosomes and functioning human genes. Proc. Natl Acad. Sci. USA 58, 1104?1111 (1967). 28. Ruddle, F. H. et al. Linkage relationships of seventeen human gene loci as determined by man?mouse somatic cell hybrids. Nature New Biol. 232, 69?73 (1971). 29. Budarf, M. L. et al. Regional localization of over 300 loci on human chromosome 22 using a somatic cell hybrid mapping panel. Genomics 35, 275?288 (1996). 30. Cox, D. R., Burmeister, M., Price, E. R., Kim, S. & Myers, R. M. Radiation hybrid mapping: a somatic cell genetic method for constructing high-resolution maps of mammalian chromosomes. Science 250, 245?250 (1990). 31. Carrano, A. V., Gray, J. W., Langlois, R. G., Burkhart- Schultz, K. J. & Van Dilla, M. A. Measurement and purification of human chromosomes by flow cytometry and sorting. Proc. Natl Acad. Sci. USA 76, 1382?1384 (1979). 32. Langlois, R. G., Yu, L. C., Gray, J. W. & Carrano, A. V. Quantitative karyotyping of human chromosomes by dual beam flow cytometry. Proc. Natl Acad. Sci. USA 79, 7876?7880 (1982). 33. Trask, B., van den Engh, G., Mayall, B. & Gray, J. W. Chromosome heteromorphism quantified by high- resolution bivariate flow karyotyping. Am. J. Hum. Genet. 45, 739?752 (1989). 34. Trask, B., van den Engh, G., Nussbaum, R., Schwartz, C. & Gray, J. Quantification of the DNA content of structurally abnormal X chromosomes and X chromosome aneuploidy using high resolution bivariate flow karyotyping. Cytometry 11, 184?195 (1990). 35. Lebo, R. V. Chromosome sorting and DNA sequence localization. Cytometry 3, 145?154 (1982). 36. Krumlauf, R., Jeanpierre, M. & Young, B. D. Construction and characterization of genomic libraries from specific human chromosomes. Proc. Natl Acad. Sci. USA 79, 2971?2975 (1982). 37. Van Dilla, M. A. & Deaven, L. L. Construction of gene libraries for each human chromosome. Cytometry 11, 208?218 (1990). 38. Telenius, H. et al. Cytogenetic analysis by chromosome painting using DOP-PCR amplified flow-sorted chromosomes. Genes Chromosomes Cancer 4, 257?263 (1992). 39. Mefford, H. C., Linardopoulou, E., Coil, D., van den Engh, G. & Trask, B. J. Comparative sequencing of a multicopy subtelomeric region containing olfactory receptor genes reveals multiple interactions between non-homologous chromosomes. Hum. Mol. Genet. 10, 2363?2372 (2001). 40. John, H. A., Birnstiel, M. L. & Jones, K. W. RNA?DNA hybrids at the cytological level. Nature 223, 582?587 (1969). � 2002 Nature Publishing Group 778 | OCTOBER 2002 | VOLUME 3 www.nature.com/reviews/genetics REVIEWS 41. Pardue, M. L. & Gall, J. G. Molecular hybridization of radioactive DNA to the DNA of cytological preparations. Proc. Natl Acad. Sci. USA 64, 600?604 (1969). 42. Langer-Safer, P. R., Levine, M. & Ward, D. C. Immunological method for mapping genes on Drosophila polytene chromosomes. Proc. Natl Acad. Sci. USA 79, 4381?4385 (1982). 43. Van Prooijen-Knegt, A. C. et al. In situ hybridization of DNA sequences in human metaphase chromosomes visualized by an indirect fluorescent immunocytochemical procedure. Exp. Cell Res. 141, 397?407 (1982). 44. Landegent, J. E. et al. Chromosomal localization of a unique gene by non-autoradiographic in situ hybridization. Nature 317, 175?177 (1985). This report shows, for the first time, the localization of a human gene to chromosome bands by non-isotopic techniques. 45. Korenberg, J. R., Chen, X. N., Adams, M. D. & Venter, J. C. Toward a cDNA map of the human genome. Genomics 29, 364?370 (1995). 46. Landegent, J. E., Jansen in de Wal, N., Dirks, R. W., Baao, F. & van der Ploeg, M. Use of whole cosmid cloned genomic sequences for chromosomal localization by non-radioactive in situ hybridization. Hum. Genet. 77, 366?370 (1987). 47. Lansdorp, P. M. et al. Heterogeneity in telomere length of human chromosomes. Hum. Mol. Genet. 5, 685?691 (1996). 48. Meyne, J. & Goodwin, E. H. Direction of DNA sequences within chromatids determined using strand-specific FISH. Chromosome Res. 3, 375?378 (1995). 49. Bailey, S. M., Meyne, J., Cornforth, M. N., McConnell, T. S. & Goodwin, E. H. A new method for detecting pericentric inversions using COD-FISH. Cytogenet. Cell Genet. 75, 248?253 (1996). 50. Cornforth, M. N. & Eberle, R. L. Termini of human chromosomes display elevated rates of mitotic recombination. Mutagenesis 16, 85?89 (2001). 51. Trask, B. J. Fluorescence in situ hybridization: applications in cytogenetics and gene mapping. Trends Genet. 7, 149?154 (1991). 52. Lichter, P. et al. High-resolution mapping of human chromosome 11 by in situ hybridization with cosmid clones. Science 247, 64?69 (1990). 53. BAC Resource Consortium. Integration of cytogenetic landmarks into the draft sequence of the human genome. Nature 409, 953?958 (2001). This paper reports the assembly and application of more than 8,000 FISH-mapped, sequence-tagged BACs, which tightly integrate the cytogenetic and sequence maps in the human genome. 54. Liu, P. et al. Fusion between transcription factor CBF?/PEBP2? and a myosin heavy chain in acute myeloid leukemia. Science 261, 1041?1044 (1993). 55. Kundu, M. & Liu, P. P. Function of the inv(16) fusion gene CBFB?MYH11. Curr. Opin. Hematol. 8, 201?205 (2001). 56. Knoll, J. H. et al. Angelman and Prader?Willi syndromes share a common chromosome 15 deletion but differ in parental origin of the deletion. Am. J. Med. Genet. 32, 285?290 (1989). 57. Lalande, M. Parental imprinting and human disease. Annu. Rev. Genet. 30, 173?195 (1996). 58. Stankiewicz, P. & Lupski, J. R. Genome architecture, rearrangements and genomic disorders. Trends Genet. 18, 74?82 (2002). 59. Cremer, T. et al. Detection of chromosome aberrations in the human interphase nucleus by visualization of specific target DNAs with radioactive and non-radioactive in situ hybridization techniques: diagnosis of trisomy 18 with probe L1.84. Hum. Genet. 74, 346?352 (1986). 60. Pinkel, D., Straume, T. & Gray, J. W. Cytogenetic analysis using quantitative, high-sensitivity, fluorescence hybridization. Proc. Natl Acad. Sci. USA 83, 2934?2938 (1986). 61. Lupski, J. R. et al. DNA duplication associated with Charcot?Marie?Tooth disease type 1A. Cell 66, 219?232 (1991). 62. Tkachuk, D. C. et al. Detection of bcr?abl fusion in chronic myelogeneous leukemia by in situ hybridization. Science 250, 559?562 (1990). This paper reports the identification of the Philadelphia chromosome in interphase nuclei using two-colour FISH. 63. Selig, S., Okumura, K., Ward, D. C. & Cedar, H. Delineation of DNA replication time zones by fluorescence in situ hybridization. EMBO J. 11, 1217?1225 (1992). 64. Kitsberg, D. et al. Allele-specific replication timing of imprinted gene regions. Nature 364, 459?463 (1993). 65. van den Engh, G., Sachs, R. & Trask, B. J. Estimating genomic distance from DNA sequence location in cell nuclei by a random walk model. Science 257, 1410?1412 (1992). 66. Wiegant, J. et al. High-resolution in situ hybridization using DNA halo preparations. Hum. Mol. Genet. 1, 587?591 (1992). 67. Parra, I. & Windle, B. High resolution visual mapping of stretched DNA by fluorescent hybridization. Nature Genet. 5, 17?21 (1993). 68. Kuwano, A., Ledbetter, S. A., Dobyns, W. B., Emanuel, B. S. & Ledbetter, D. H. Detection of deletions and cryptic translocations in Miller?Dieker syndrome by in situ hybridization. Am. J. Hum. Genet. 49, 707?714 (1991). 69. Schrock, E. et al. Multicolor spectral karyotyping of human chromosomes. Science 273, 494?497 (1996). This paper and reference 70 show how each human chromosome can be painted with one of 24 colours for automated karyotype analysis. 70. Speicher, M. R., Gwyn Ballard, S. & Ward, D. C. Karyotyping human chromosomes by combinatorial multi- fluor FISH. Nature Genet. 12, 368?375 (1996). 71. Lichter, P. et al. Rapid detection of human chromosome 21 aberrations by in situ hybridization. Proc. Natl Acad. Sci. USA 85, 9664?9668 (1988). 72. Pinkel, D. et al. Fluorescence in situ hybridization with human chromosome-specific libraries: detection of trisomy 21 and translocations of chromosome 4. Proc. Natl Acad. Sci. USA 85, 9138?9142 (1988). 73. Vooijs, M. et al. Libraries for each human chromosome, constructed from sorter-enriched chromosomes by using linker?adaptor PCR. Am. J. Hum. Genet. 52, 586?597 (1993). 74. Meltzer, P. S., Guan, X. Y., Burgess, A. & Trent, J. M. Rapid generation of region specific probes by chromosome microdissection and their application. Nature Genet. 1, 24?28 (1992). 75. Ried, T., Landes, G., Dackowski, W., Klinger, K. & Ward, D. C. Multicolor fluorescence in situ hybridization for the simultaneous detection of probe sets for chromosomes 13, 18, 21, X and Y in uncultured amniotic fluid cells. Hum. Mol. Genet. 1, 307?313 (1992). 76. Mrozek, K., Heinonen, K., Theil, K. S. & Bloomfield, C. D. Spectral karyotyping in patients with acute myeloid leukemia and a complex karyotype shows hidden aberrations, including recurrent overrepresentation of 21q, 11q, and 22q. Genes Chromosomes Cancer 34, 137?153 (2002). 77. Liyanage, M. et al. Multicolour spectral karyotyping of mouse chromosomes. Nature Genet. 14, 312?315 (1996). 78. Loucas, B. D. & Cornforth, M. N. Complex chromosome exchanges induced by ?-rays in human lymphocytes: an mFISH study. Radiat. Res. 155, 660?671 (2001). 79. Sachs, R. K., Hlatky, L. R. & Trask, B. J. Radiation- produced chromosome aberrations: colourful clues. Trends Genet. 16, 143?146 (2000). 80. Knight, S. J. et al. An optimized set of human telomere clones for studying telomere integrity and architecture. Am. J. Hum. Genet. 67, 320?332 (2000). 81. Flint, J. et al. The detection of subtelomeric chromosomal rearrangements in idiopathic mental retardation. Nature Genet. 9, 132?140 (1995). 82. Jauch, A. et al. Reconstruction of genomic rearrangements in great apes and gibbons by chromosome painting. Proc. Natl Acad. Sci. USA 89, 8611?8615 (1992). 83. Weinberg, J. & Stanyon, R. Comparative painting of mammalian chromosomes. Curr. Opin. Genet. Dev. 7, 784?791 (1997). A review of the chromosomal rearrangements that have occurred during evolution as detected by cross- species FISH using chromosome-specific paints and locus-specific probes. 84. Stanyon, R. et al. Reciprocal chromosome painting shows that genomic rearrangement between rat and mouse proceeds ten times faster than between humans and cats. Cytogenet. Cell. Genet. 84, 150?155 (1999). 85. Ostrander, E. A. & Kruglyak, L. Unleashing the canine genome. Genome Res. 10, 1271?1274 (2000). 86. Breen, M., Thomas, R., Binns, M. M., Carter, N. P. & Langford, C. F. Reciprocal chromosome painting reveals detailed regions of conserved synteny between the karyotypes of the domestic dog (Canis familiaris) and human. Genomics 61, 145?155 (1999). 87. Kallioniemi, A. et al. Comparative genomic hybridization for molecular cytogenetic analysis of solid tumors. Science 258, 818?821 (1992). The first paper to describe CGH, which makes it possible to detect loss and gain of chromosomal material in non-dividing tumour cells. 88. Shayesteh, L. et al. PIK3CA is implicated as an oncogene in ovarian cancer. Nature Genet. 21, 99?102 (1999). 89. Klein, C. A. et al. Comparative genomic hybridization, loss of heterozygosity, and DNA sequence analysis of single cells. Proc. Natl Acad. Sci. USA 96, 4494?4499 (1999). 90. Pinkel, D. et al. High resolution analysis of DNA copy number variation using comparative genomic hybridization to microarrays. Nature Genet. 20, 207?211 (1998). Provides the first proof-of-principle demonstration of array-CGH using BAC clones that were selected to mark specific points along the genome as hybridization targets. 91. Schriml, L. M. et al. Tyramide signal amplification (TSA)- FISH applied to mapping PCR-labeled probes less than 1 kb in size. Biotechniques 27, 608?613 (1999). 92. Zhong, X. B., Lizardi, P. M., Huang, X. H., Bray-Ward, P. L. & Ward, D. C. Visualization of oligonucleotide probes and point mutations in interphase nuclei and DNA fibers using rolling circle DNA amplification. Proc. Natl Acad. Sci. USA 98, 3940?3945 (2001). 93. Suzuki, H. et al. A genomic screen for genes upregulated by demethylation and histone deacetylase inhibition in human colorectal cancer. Nature Genet. 31, 141?149 (2002). 94. Iyer, V. R. et al. Genomic binding sites of the yeast cell- cycle transcription factors SBF and MBF. Nature 409, 533?538 (2001). 95. Gray, J. W. et al. Flow karyotyping and sorting of human chromosomes. Cold Spring Harbor Symp. Quant. Biol. 51, 141?149 (1986). 96. Zitzelsberger, H. F., O?Brien, B. & Weier, H. U. G. in FISH Technology (eds Rautenstrauss, B. & Liehr, T.) 408?424 (Springer, Heidelberg, 2002). 97. McNeil, N. & Ried, T. Novel molecular cytogenetic techniques for identifying complex chromosomal rearrangements: technology and applications in molecular medicine. Expert Rev. Mol. Med. [online] 14 September 2000 <http://www-ermm.cbcu.cam.ac.uk/ 00001940h.htm> (2000). 98. Padilla-Nash, H. M. et al. Molecular cytogenetic analysis of the bladder carcinoma cell line BK-10 by spectral karyotyping. Genes Chromosomes Cancer 25, 53?59 (1999). Acknowledgements I would like to thank the National Institutes of Health, US Department of Energy and the Fred Hutchinson Cancer Research Center for their present and past support. B.J.T. has significant financial interest in Cytopeia and Dako Cytomation, which are com- panies that develop and market flow cytometers. Online links DATABASES The following terms in this article are linked online to: LocusLink: http://www.ncbi.nlm.nih.gov/LocusLink ABL | BCR | CBFB | HOXA9 | MYH11 | NUP98 | PIK3CA | RB | thyroglobulin OMIM: http://www.ncbi.nlm.nih.gov/Omim acute myelogenous leukaemia | Angelman syndrome | Charcot?Marie?Tooth syndrome | chronic myeloid leukaemia | Cri-du-Chat syndrome | Down syndrome | Klinefelter syndrome | Prader?Willi syndrome | retinoblastoma | Turner syndrome FURTHER INFORMATION Atlas of Genetics and Cytogenetics in Oncology and Haematology: http://www.infobiogen.fr/services/chromcancer/index.html Cytogenetic Forum at Waisman Center, University of Wisconsin: http://www.waisman.wisc.edu/cytogenetics/index.htmlx Developmental Genome Anatomy Project: http://www.bwhpathology.org/dgap Mitelman Database of Chromosome Aberrations in Cancer: http://cgap.nci.nih.gov/Chromosomes/Mitelman Access to this interactive links box is free online. "
Add Content to Group
|
Bookmark
|
Keywords
|
Flag Inappropriate
share
Close
Digg
Facebook
MySpace
Google+
Comments
Close
Please Post Your Comment
*
The Comment you have entered exceeds the maximum length.
Submit
|
Cancel
*
Required
Comments
Please Post Your Comment
No comments yet.
Save Note
Note
View
Public
Private
Friends & Groups
Friends
Groups
Save
|
Cancel
|
Delete
Please provide your notes.
Next
|
Prev
|
Close
|
Edit
|
Delete
Genetics
Gene Inheritance and Transmission
Gene Expression and Regulation
Nucleic Acid Structure and Function
Chromosomes and Cytogenetics
Evolutionary Genetics
Population and Quantitative Genetics
Genomics
Genes and Disease
Genetics and Society
Cell Biology
Cell Origins and Metabolism
Proteins and Gene Expression
Subcellular Compartments
Cell Communication
Cell Cycle and Cell Division
Scientific Communication
Career Planning
Loading ...
Scitable Chat
Register
|
Sign In
Visual Browse
Close
Comments
CloseComments
Please Post Your Comment