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Detailed knowledge of chromosomal aberrations in a specific tumor may facilitate the development of individually tailored chemotherapy, hormone or gene therapy. Unfortunately, karyotype analysis requires living cells and is complicated by the low number of good metaphase spreads obtained. Comparative genomic hybridization (CGH), however, is capable of detecting and mapping genome-wide amplifications and deletions using an equimolar mixture of normal and tumor cell DNA. We show here that even the few cells from a fine needle aspirate of a tumor are sufficient for a direct CGH assay, independent of DNA amplification. Ten primary breast cancers were analyzed by CGH. A fresh frozen fine needle aspirate and a formalin-fixed and paraffin-embedded section were used for each tumor. Metaphases from each CGH reaction were imaged, and a sum ratio profile was determined for every chromosome. The ratio profiles of DNA isolated from the 2 material sources were then compared. Fine needle aspirates and the paraffin-embedded material of a single tumor yielded the same fluorescence ratio profiles, albeit with slightly different confidence intervals. Different tumors showed a variety of aberrations. The most frequently observed changes were 1q+, 8q+, 14q-, 16p+, 16q-, 17p-, 17q+, 19q+, 20q+, 21q- and 22q-. The ability of CGH to assess chromosomal changes in breast cancer from fine needle aspirates could facilitate genetic evaluation of tumors prior to surgery., (Copyright 2000 Wiley-Liss, Inc.)

Comparative genomic hybridization (CGH) is a FISH-related technique used to assess global chromosomal aberrations in a variety of human tumours. Recently CGH has been applied to cytogenetic analysis of fresh frozen fetoplacental tissues. Here we report the application of CGH to paraffin-embedded placental samples. Ten samples from paraffin-embedded blocks of 6 control placentas and fetoplacental tissue from 10 aneuploidies, and 2 unbalanced aberrations were evaluated. Balanced karyotype profiles were obtained from samples of healthy placentas and all samples from the same placenta appeared to have similar confidence intervals. CGH analysis of four cases of trisomy 21, three cases of trisomy 18, one case of trisomy 13, one case of trisomy 15 and one case of trisomy 7 all showed overrepresentation of the respective trisomic chromosome. The CGH profile was also in accordance with the karyotyping of a case with isochromosome 21. The CGH profile of a case with der (2)t(2;6)(q37.3;q22.2) revealed partial trisomy for chromosome 6 between q21 and q27. CGH may be a useful adjunct in prenatal genetic diagnosis when retrospective diagnosis is needed from archival samples.

Fluorescence in situ hybridization has been used to visualize specific genomic DNA sequences in interphase nuclei. In normal diploid cells, unreplicated DNA segments give singlet hybridization signals while replicated loci are characterized by doublets. The distribution of these two patterns in unsynchronized cell populations can be used to determine the S phase replication time of any DNA sequence. The validity of this approach was established by analyzing genes whose replication profiles in expressing and non-expressing cells had been determined previously by conventional methods. Using this technique it has been possible to map the replication timing topography of the DNA within and flanking the cystic fibrosis (CF) gene locus on chromosome 7. The gene itself is located within a defined time zone which is approximately 500 kb in length and is under developmental control. It is early replicating in cells which express CF but late replicating in other cell types. These time zones probably represent basic units of chromosome structure.

Nonisotopic in situ hybridization is a powerful tool to analyze the organization of complex genomes. Current approaches utilizing this technique for the analysis of linear and spatial genome organizations are presented. Clinical applications of these approaches, which open new avenues for diagnosis of disease-related chromosomal changes, are also discussed.

We developed a method for the biopsy of preimplantation mouse embryos (preembryos) at the four- to eight-cell stage, which uses partial zona pellucida dissection. The preembryos were collected in calcium- and magnesium-free phosphate-buffered saline solution with 0.01% ethylenediaminetetraacetic acid, 0.1 mol/L sucrose, and 4 mg/ml of bovine serum albumin to facilitate removal of blastomeres. This allows entry of a fine micropipette into the perivitelline cavity with subsequent removal of a single blastomere by gentle suction. The majority of embryos (75%) from which biopsy specimens were obtained in this fashion developed to the blastocyst stage. The blastomeres obtained were mainly intact and they were fixed to glass slides. After permeabilization, in situ hybridization was performed with chromosome X- and chromosome 3-specific probes. Human unfertilized eggs and blastomeres from human polyspermic embryos also have been analyzed by in situ hybridization with chromosome specific probes. The combination of nondestructive embryo biopsy and in situ hybridization is a possible approach for preimplantation genetic diagnosis.

Cosmid clones containing human DNA inserts have been mapped on chromosome 11 by fluorescence in situ hybridization under conditions that suppress signal from repetitive DNA sequences. Thirteen known genes, one chromosome 11-specific DNA repeat, and 36 random clones were analyzed. High-resolution mapping was facilitated by using digital imaging microscopy and by analyzing extended (prometaphase) chromosomes. The map coordinates established by in situ hybridization showed a one to one correspondence with those determined by Southern (DNA) blot analysis of hybrid cell lines containing fragments of chromosome 11. Furthermore, by hybridizing three or more cosmids simultaneously, gene order on the chromosome could be established unequivocally. These results demonstrate the feasibility of rapidly producing high-resolution maps of human chromosomes by in situ hybridization.

We have developed a novel method for high resolution mapping of specific DNA sequences after in situ hybridization. DNA probes, labeled with biotin-nucleotides in conventional nick-translation reactions, are hybridized to cytological preparations and detected with affinity-purified rabbit antibiotin antibodies followed by antibodies to rabbit IgG that are conjugated to fluorescent or enzymatic reagents. Using peroxidase labeled anti-rabbit IgG, we are able to detect and localize specific sequences at both the light and electron microscopic levels. Initial studies were done with repeated DNA sequences previously mapped by light microscope autoradiography to assess the fidelity and resolution of this method. An analysis using biotin-labeled mouse satellite DNA is presented here.

A method is described for localizing DNA sequences hybridized in situ to Drosophila polytene chromosomes. This procedure utilizes a biotin-labeled analog of TTP that can be incorporated enzymatically into DNA probes by nick-translation. After hybridization in situ, the biotin molecules in the probe serve as antigens which bind affinity-purified rabbit antibiotin antibodies. The site of hybridization is then detected either fluorimetrically, by using fluorescein-labeled goat anti-rabbit IgG, or cytochemically, by using an anti-rabbit IgG antibody conjugated to horseradish peroxidase. When combined with Giemsa staining, the immunoperoxidase detection method provides a permanent record that is suitable for detailed cytogenetic analysis. This immunological approach offers four advantages over conventional autoradiographic procedures for detecting in situ hybrids: (i) the time required to determine the site of hybridization is decreased markedly, (ii) biotin-labeled probes are chemically stable and give reproducible results for many months; (iii) biotin-labeled probes appear to produce less background noise than do radiolabeled probes; and (iv) the resolving power is equal to and often greater than that achieved autoradiographically.

Biotin-labelled DNA probes, prepared by nick-translation in the presence of biotinylated analogs of TTP, are hybridized to DNA or RNA immobilized on nitrocellulose filters. After removal of residual probe, the filters are incubated for 2--5 min with a preformed complex made with avidin-DH (or streptavidin) and biotinylated polymers of intestinal alkaline phosphatase. The filters are then incubated with a mixture of 5-bromo-4-chloro-3-indolyl phosphate and nitro blue tetrazolium, which results in the deposition of a purple precipitate at the sites of hybridization. This procedure will detect target sequences in the 1- to 10-pg range after enzyme incubation periods of 1 hr or less. The incubation period can be extended up to 24 hr, if required, to increase the color intensity of the hybridization signal. Furthermore, at high probe concentrations (250--7560 ng/ml), biotin-labeled DNA exhibits lower nonspecific binding to nitrocellulose than does radiolabeled DNA, so hybridization times required for the analysis of unique mammalian gene sequences can be decreased to 1--2 hr. This nonradiographic method of probe detection should be of general utility for genetic studies using Southern, RNA, or dot-blot hybridization protocols.

Chromosome aberrations in two glioma cell lines were analyzed using biotinylated DNA library probes that specifically decorate chromosomes 1, 4, 7, 18 and 22 from pter to qter. Numerical changes, deletions and rearrangements of these chromosomes were readily visualized in metaphase spreads, as well as in early prophase and interphase nuclei. Complete chromosomes, deleted chromosomes and segments of translocated chromosomes were rapidly delineated in very complex karyotypes. Simultaneous hybridizations with additional subregional probes were used to further define aberrant chromosomes. Digital image analysis was used to quantitate the total complement of specific chromosomal DNAs in individual metaphase and interphase cells of each cell line. In spite of the fact that both glioma lines have been passaged in vitro for many years, an under-representation of chromosome 22 and an over-representation of chromosome 7 (specifically 7p) were observed. These observations agree with previous studies on gliomas. In addition, sequences of chromosome 4 were also found to be under-represented, especially in TC 593. These analyses indicate the power of these methods for pinpointing chromosome segments that are altered in specific types of tumors.

A molecular hybridization technique was developed to detect bunyavirus RNA in cells. Complementary DNAs (cDNAs) to the small (S) RNA segment of La Crosse (LAC) virus and to a portion of the middle (M) RNA segment of snowshoe hare (SSH) virus were used as probes to detect LAC or SSH viral RNA by in situ hybridization. Protocols were developed and standardized using radiolabeled DNA probes, and adapted for use with biotin labeled probes. The in situ hybridization procedure detected an estimated 3,600 copies of viral S RNA/cell at 24 hr postinfection. In growth curve studies, LAC nucleocapsid antigen was detectable slightly before S RNA. LAC S RNA synthesis was first seen about the nucleus. By 12 hr postinfection, hybridization signal was detected throughout the cytoplasm of the cell. The LAC S RNA probe was group-specific and cross-hybridized to 5 other California group viruses. The SSH M RNA probe was type-specific.

A method for the rapid physical isolation of recombinant plasmids of interest from a mixture of plasmids such as a plasmid cDNA library is presented. This method utilizes the ability of RecA protein to form stable complexes between linear single-stranded and circular double-stranded DNA molecules sharing sequence homology, and procedures allowing isolation of biotinylated nucleic acid. Biotinylated linear DNA probes coated with RecA have been used to screen reconstituted plasmid libraries consisting of two plasmid species, one homologous and the other heterologous to the probe. When the link between biotin and the nucleotide base could be cleaved by reducing agents, the complex was purified by streptavidin-agarose chromatography and the recovered plasmid was propagated in Escherichia coli. When the link was not cleavable the complex was bound to avidin in solution and purified by cupric iminodiacetic acid-agarose chromatography. The complex was then dissociated and the plasmids were propagated in E. coli. With either protocol, homologous plasmid recovery was between 10% and 20%, and enrichment was between 10(4)- and 10(5)-fold. Potential applications and extensions of this method, such as plasmid, cosmid, and phage library screening and facilitation of physical mapping of macroregions of mammalian genomes are presented and discussed.

In situ hybridization has become a standard method for localizing DNA or RNA sequences in cytological preparations. We developed two methods to extend this technique to the transmission electron microscope level using mouse satellite DNA hybridization to whole mount metaphase chromosomes as the test system. The first method devised is a direct extension of standard light microscope level using mouse satellite DNA hybridization to whole mount metaphase chromosomes as the test system. The first method devised is a direct extension of standard light microscope in situ hybridization. Radioactively labeled complementary RNA (cRNA) is hybridized to metaphase chromosomes deposited on electron microscope grids and fixed in 70 percent ethanol vapor; hybridixation site are detected by autoradiography. Specific and intense labeling of chromosomal centromeric regions is observed even after relatively short exposure times. Inerphase nuclei present in some of the metaphase chromosome preparations also show defined paatterms of satellite DNA labeling which suggests that satellite-containing regions are associate with each other during interphase. The sensitivity of this method is estimated to at least as good as that at the light microscope level while the resolution is improved at least threefold. The second method, which circumvents the use of autoradiogrphic detection, uses biotin-labeled polynucleotide probes. After hybridization of these probes, either DNA or RNA, to fixed chromosomes on grids, hybrids are detected via reaction is improved at least threefold. The second method, which circumvents the use of autoradiographic detection, uses biotin-labeled polynucleotide probes. After hybridization of these probes, either DNA or RNA, to fixed chromosomes on grids, hybrids are detected via reaction with an antibody against biotin and secondary antibody adsorbed to the surface of over centromeric heterochromatin and along the associated peripheral fibers. Labeling is on average ten times that of background binding. This method is rapid and possesses the potential to allow precise ultrastructual localization of DNA sequences in chromosomes and chromatin.

A method of in situ hybridization for visualizing individual human chromosomes from pter to qter, both in metaphase spreads and interphase nuclei, is reported. DNA inserts from a single chromosomal library are labeled with biotin and partially preannealed with a titrated amount of total human genomic DNA prior to hybridization with cellular or chromosomal preparations. The cross-hybridization of repetitive sequences to nontargeted chromosomes can be markedly suppressed under appropriate preannealing conditions. The remaining single-stranded DNA is hybridized to specimens of interest and detected with fluorescent or enzyme-labeled avidin conjugates following post-hybridization washes. DNA inserts from recombinant libraries for chromosomes 1, 4, 7, 8, 13, 14, 18, 20, 21, 22, and X were assessed for their ability to decorate specifically their cognate chromosome; most libraries proved to be highly specific. Quantitative densitometric analyses indicated that the ratio of specific to nonspecific hybridization signal under optimal preannealing conditions was at least 8:1. Interphase nuclei showed a cohesive territorial organization of chromosomal domains, and laser-scanning confocal fluorescence microscopy was used to aid the 3-D visualization of these domains. This method should be useful for both karyotypic studies and for the analysis of chromosome topography in interphase cells.

A method of in situ cytohybridization is described for the detection of specific viral genomes in infected cell cultures or paraffin-embedded tissue sections without the use of radioisotopes. Biotin-labeled analogs of TTP are incorporated into viral DNA in vitro by nick translation and the resultant DNA probes hybridized to cytologic samples. Cells containing viral genetic material are then revealed by standard immunofluorescence, immunoperoxidase, or affinity cytochemical techniques that are based on the specific interaction between biotin and antibiotin IgG or avidin. Hybridization probes containing nucleotides that have an 11- or 16-atom spacer arm between the biotin molecule and the pyrimidine ring interact with these detector proteins more efficiently than probes containing biotin-nucleotides with a 4-atom spacer arm. The total procedure can be performed fairly rapidly (24 hr or less) and numerous samples can be processed simultaneously. Although the detection methods employed to date are not as sensitive as autoradiographic procedures with high specific activity probes, more sensitive protein detector complexes are currently being constructed. The speed, specificity, and resolving power of this technique should be of general utility in screening for the presence of infectious agents in cell or tissue samples. Here we report the visualization of parvovirus, polyomavirus, herpes simplex virus, adenovirus, and retrovirus genetic material in infected cell cultures and herpes simplex and adenovirus DNA in paraffin-embedded autopsy tissues.