High-resolution array comparative genomic hybridization of single micrometastatic tumor cells

Only few selected cancer cells drive tumor progression and are responsible for therapy resistance. Their specific genomic characteristics, however, are largely unknown because high-resolution genome analysis is currently limited to DNA pooled from many cells. Here, we describe a protocol for array comparative genomic hybridization (array CGH), which enables the detection of DNA copy number changes in single cells. Combining a PCR-based whole genome amplification method with arrays of highly purified BAC clones we could accurately determine known chromosomal changes such as trisomy 21 in single leukocytes as well as complex genomic imbalances of single cell line cells. In single T47D cells aberrant regions as small as 1–2 Mb were identified in most cases when compared to non-amplified DNA from 10⁶ cells. Most importantly, in single micrometastatic cancer cells isolated from bone marrow of breast cancer patients, we retrieved and confirmed amplifications as small as 4.4 and 5 Mb. Thus, high-resolution genome analysis of single metastatic precursor cells is now possible and may be used for the identification of novel therapy target genes.     

Cytogenetic analysis from DNA by comparative genomic hybridization

Comparative genomic hybridization (CGH) is a modified in situ hybridization technique which allows detection and mapping of DNA sequence copy differences between two genomes in a single experiment. In CGH analysis, two differentially labelled genomic DNA (study and reference) are co-hybridized to normal metaphase spreads. Chromosomal locations of copy number changes in the DNA segments of the study genome are revealed by a variable fluorescence intensity ratio along each target chromosome. Since its development, CGH has been applied mostly as a research tool in the field of cancer cytogenetics to identify genetic changes in many previously unknown regions. CGH may also have a role in clinical cytogenetics for detection and identification of unbalanced chromosomal abnormalities.     

High-resolution in situ hybridization to whole-mount zebrafish embryos

The in situ hybridization (ISH) technique allows the sites of expression of particular genes to be detected. This protocol describes ISH of digoxigenin-labeled antisense RNA probes to whole-mount zebrafish embryos. In our method, PCR-amplified sequence of a gene of interest is used as a template for the synthesis of an antisense RNA probe, which is labeled with digoxigenin-linked nucleotides. Embryos are fixed and permeabilized before being soaked in the digoxigenin-labeled probe. We use conditions that favor specific hybridization to complementary mRNA sequences in the tissue(s) expressing the corresponding gene. After washing away excess probe, hybrids are detected by immunohistochemistry using an alkaline phosphatase-conjugated antibody against digoxigenin and a chromogenic substrate. The whole procedure takes only 3 days and, because ISH conditions are the same for each probe tested, allows high throughput analysis of zebrafish gene expression during embryogenesis.     

High-resolution mapping of genotype-phenotype relationships in cri du chat syndrome using array comparative genomic hybridization

We have used array comparative genomic hybridization to map DNA copy-number changes in 94 patients with cri du chat syndrome who had been carefully evaluated for the presence of the characteristic cry, speech delay, facial dysmorphology, and level of mental retardation (MR). Most subjects had simple deletions involving 5p (67 terminal and 12 interstitial). Genotype-phenotype correlations localized the region associated with the cry to 1.5 Mb in distal 5p15.31, between bacterial artificial chromosomes (BACs) containing markers D5S2054 and D5S676; speech delay to 3.2 Mb in 5p15.32-15.33, between BACs containing D5S417 and D5S635; and the region associated with facial dysmorphology to 2.4 Mb in 5p15.2-15.31, between BACs containing D5S208 and D5S2887. These results overlap and refine those reported in previous publications. MR depended approximately on the 5p deletion size and location, but there were many cases in which the retardation was disproportionately severe, given the 5p deletion. All 15 of these cases, approximately two-thirds of the severely retarded patients, were found to have copy-number aberrations in addition to the 5p deletion. Restriction of consideration to patients with only 5p deletions clarified the effect of such deletions and suggested the presence of three regions, MRI-III, with differing effect on retardation. Deletions including MRI, a 1.2-Mb region overlapping the previously defined cri du chat critical region but not including MRII and MRIII, produced a moderate level of retardation. Deletions restricted to MRII, located just proximal to MRI, produced a milder level of retardation, whereas deletions restricted to the still-more proximal MRIII produced no discernible phenotype. However, MR increased as deletions that included MRI extended progressively into MRII and MRIII, and MR became profound when all three regions were deleted.     

High-resolution physical mapping in Arabidopsis thaliana and tomato by fluorescence in situ hybridization to extended DNA fibres

A technique to detect DNA sequences on extended DNA fibres (EDF) prepared from interphase nuclei from tomato (Lycopersicon esculentum) and Arabidopsis thaliana leaves by fluorescence in situ hybridization (FISH) is described. Three nuclear lysis procedures have been tested for their ability to decondense chromatin and to generate highly extended intact DNA fibres on microscopic slides. DNA probes of various sizes have been used in FISH experiments to EDFs to establish the resolution and sensitivity of the technique. The fluorescent signals of a 5S rDNA probe hybridized to tomato EDFs revealed continuous strings of about 200 µm, that corresponded to a molecular size of about 660 kb. In A. thaliana, a contig of three cosmids spanning a genomic region with a total length of about 89 kb was analysed. By means of multi-colour hybridization the physical positions of the cosmids were visualized as red and green fluorescence strings with overlapping regions in yellow. Comparison of the length of the fluorescent signals with the molecular data revealed a stretching degree of the DNA fibres at 3.27 kb µm^?1, which is close to the Watson-Crick DNA length estimate of 2.9 kb µm^?1. Other experiments on small size molecular probes with both lambda clones (13.5–17 kb insert sizes) and plasmids (4.2 and 5 kb) in a contig of A. thaliana, and the 5S rDNA region in tomato showed close agreement with molecular data. The lower limit of the detection, which was established in a hybridization experiment with two DNA probes from the 45S ribosomal gene on extended fibres of tomato, was about 0.7 kb. Consistent patterns of alternating fluorescent red and green spots were obtained reflecting the tandemly repeated arrangement of the 18S and 25S ribosomal sequences. On the basis of the microscopic distance between these hybridization spots the size of the ribosomal unit was estimated at 8.2 kb. This implies a drastic improvement of high-resolution physical mapping of DNA sequences by FISH on plant DNA.     

High-resolution physical mapping of the secalin-1 locus of rye on extended DNA fibers

High-resolution mapping of secalin-1 (Sec-1) locus has been performed by fluorescence in situ hybridization to extended DNA fibers of rye (Secale cereale, 2n = 14), employing DNA probes of lambda phage clones containing the omega-secalin gene. The fluorescent signals to rye extended DNA fibers revealed continuous strings of 45 microm, corresponding to the size of 147 kb DNA. To determine the copy number of Sec-1 locus on DNA fibers, a 1.2-kb fragment including the entire coding region of the omega-secalin gene and a 1.0-kb fragment of the promoter region were amplified by PCR as probes for another fiber FISH. The physical position of these sequences was visualized as alternating fluorescent spots by multicolor in situ hybridization. Alternating signals of two DNA probes reflected the tandem repeated organization of the Sec-1 locus having 15 copies of the gene. The present findings based on fiber FISH analysis support the contention that the omega-secalin genes are arranged in a head-to-tail fashion separated by 8 kb of spacer sequences with a total length of 145 kb.     

Fluorescence in situ hybridization on plant extended chromatin DNA fibers for single-copy and repetitive DNA sequences

The compactness of plant chromosomes and the structure of the plant cell wall and cytoplasm provide a great obstacle to fluorescence in situ hybridization (FISH) for single-copy or low-copy DNA sequences. Consequently, many new methods for improving spatial resolution via chromosomal stretching have been employed to overcome this technical challenge. In this article, a technique for extracting cell-wall free nuclei at mitotic interphase, then using these nuclei to prepare extended DNA fibers (EDFs) by the method of a receding interface, whereby slide-mounted chromatin produces EDFs in concert with gravity-assisted buffer flow, was adopted as a result of the low frequency of EDF damage produced by this procedure. To examine the quality of these EDFs, we used single-copy gene encoding S-locus receptor kinase and multi-copy 5S rDNA (ribosomal DNA) as probes. The resulting EDFs proved suitable for high-resolution FISH mapping for repetitive DNA sequences, and the localization of a single-copy locus.     

Fast combinatorial cartography by FISH on combed genomic DNA

A novel combinatorial method combining FISH on combed genomic DNA is presented for a fast high-resolution (1Kbps) gene cartography.This revised version was published online in October 2005 with corrections to the Cover Date.     

Optimization of DOP-PCR amplification of DNA for high-resolution comparative genomic hybridization analysis

BACKGROUND: Whole-genome amplification of minute samples of DNA for the use in comparative genomic hybridization (CGH) analysis has found widespread use, but the method has not been well validated. METHODS: Four protocols for degenerate oligonucleotide primed polymerase chain reaction (DOP-PCR) and fluorescence labeling were applied to test DNA from normal and K-562 cells. The DNA products were used for CGH analysis. RESULTS: The DOP-PCR-amplified DNA from each protocol produced hybridizations with different qualities. These could be seen primarily as differences in background staining and signal-to-noise ratios, but also as characteristic deviations of normal/normal hybridizations. One DOP-PCR-protocol was further investigated. We observed concordance between CGH results using unamplified and DOP-PCR-amplified DNA. An example of an analysis of an invasive carcinoma of the breast supports the practical value of this approach. CONCLUSIONS: DOP-PCR-amplified DNA is applicable for high- resolution CGH, the results being similar to those of CGH using unamplified DNA.     

Detection of DNA copy number changes in human endometriosis by comparative genomic hybridization

Endometriosis is characterized by infertility and pelvic pain in 10-15% of women of reproductive age. The genetic events involved in endometriotic cell expansion remain in large part unknown. To identify genomic changes involved in development of this disease, we examined a panel of 18 selected endometriotic tissues by comparative genomic hybridization (CGH), a molecular cytogenetic method that allows screening of the entire genome for chromosomal gains and/or losses. The study was performed on native, nonamplified DNA extracted from manually dissected endometriotic lesions. Recurrent copy number losses on several chromosomes were detected in 15 of 18 cases. Loss of chromosome 1p and 22q were detected in 50% of the cases. Additional common losses occurred on chromosomes 5p (33%), 6q (27%), 7p(22%), 9q (22%), 16 (22%) as well as on 17q in one case. Gain of DNA sequences were seen at 6q, 7q and 17q in three cases. To validate the CGH data, selective dual-color FISH was performed using probes for the deleted regions on chromosomes 1, 7 and 22 in parallel with the corresponding centromeric probes. Cases showing deletion by CGH all had two signals at 1p36, 7p22.1 and 22q12 in less than 30% of the nuclei in comparison to the double centromeric labels found in more than 85% of the cells. These findings indicate that genes localized to previously undescribed chromosomal regions play a role in development and progression of endometriosis.     

High-resolution array comparative genomic hybridization of chromosome 8q: evaluation of putative progression markers for gastroesophageal junction adenocarcinomas

Amplification of 8q is frequently found in gastroesophageal junction (GEJ) cancer. It is usually detected in high-grade, high-stage GEJ adenocarcinomas. Moreover, it has been implicated in tumor progression in other cancer types. In this study, a detailed genomic analysis of 8q was performed on a series of GEJ adenocarcinomas, including 22 primary adenocarcinomas, 13 cell lines and two xenografts, by array comparative genomic hybridization (aCGH) with a whole chromosome 8q contig array. Of the 37 specimens, 21 originated from the esophagus and 16 were derived from the gastric cardia. Commonly overrepresented regions were identified at distal 8q, i.e. 124-125 Mb (8q24.13), at 127-128 Mb (8q24.21), and at 141-142 Mb (8q24.3). From these regions six genes were selected with putative relevance to cancer: ANXA13, MTSS1, FAM84B (alias NSE2), MYC, C8orf17 (alias MOST-1) and PTK2 (alias FAK). In addition, the gene EXT1 was selected since it was found in a specific amplification in cell line SK-GT-5. Quantitative RT-PCR analysis of these seven genes was subsequently performed on a panel of 24 gastroesophageal samples, including 13 cell lines, two xenografts and nine normal stomach controls. Significant overexpression was found for MYC and EXT1 in GEJ adenocarcinoma cell lines and xenografts compared to normal controls. Expression of the genes MTSS1, FAM84B and C8orf17 was found to be significantly decreased in this set of cell lines and xenografts. We conclude that, firstly, there are other genes than MYC involved in the 8q amplification in GEJ cancer. Secondly, the differential expression of these genes contributes to unravel the biology of GEJ adenocarcinomas.     

Characterization of Aegilops uniaristata chromosomes by comparative DNA marker analysis and repetitive DNA sequence in situ hybridization

RFLP analyses were performed on wheat-Aegilops uniaristata Vis. addition and translocation lines to confirm the identity of added N-genome chromosomes. Complete 1N, 3N, 4N, 5N and 7N chromosome additions were identified, while the complete long arm and only part of the short arm was identified for chromosome 2N. There were no wheat-like 4/5 and 4/7 translocations in the Ae. uniaristata chromosomes. Chromosome 3N carried an asymmetric pericentric inversion, and the translocation line was a product of centric fusion between the long arms of chromosomes 3B and 3N. Chromosome-specific RAPD and microsatellite markers were also identified for all the added Ae. uniaristata chromosomes available in this set of addition lines. A new genomic in situ hybridization protocol combining pre-annealing of probe and blocking DNA and prehybridization with blocking DNA was developed to differentiate the very closely related genomes of Ae. uniaristata and wheat. Hybridization sites for the repetitive DNA sequences pAs1, pSc119.2 and pTa71 were identified on the N-genome chromosomes of Ae. uniaristata using the fluorescent in situ hybridization technique. Results showed deviation from the previously published ideogram of this species. A new ideogram, which shows the hybridization sites for the above sequences, was produced in which the chromosomes are arranged according to their homoeologous group. Received: 23 April 1999 / Accepted: 6 August 1999     

Direct hybridization of labeled DNA to DNA in agarose gels

A naringinase assay capable of distinguishing between the content of naringin, prunin, and naringenin present in the incubation mixture, is described. The amount of these compounds can be estimated by combining two spectrophotometric procedures. (a) Treatment with strong alkali to determine the amount of nargingenin as well as the sum of naringin and prunin. (b) Assay of the liberated aldohexoses with o-aminodiphenyl. From the data thus obtained, the amount of the remaining substrate, the amount of the intermediate as well as the product at any given time can be calculated.     

An oligonucleotide hybridization approach to DNA sequencing

We have proposed a DNA sequencing method based on hybridization of a DNA fragment to be sequenced with the complete set of fixed-length oligonucleotides (e.g., 4(8) = 65,536 possible 8-mers) immobilized individually as dots of a 2-D matrix [(1989) Dokl. Akad. Nauk SSSR 303, 1508-1511]. It was shown that the list of hybridizing octanucleotides is sufficient for the computer-assisted reconstruction of the structures for 80% of random-sequence fragments up to 200 bases long, based on the analysis of the octanucleotide overlapping. Here a refinement of the method and some experimental data are presented. We have performed hybridizations with oligonucleotides immobilized on a glass plate, and obtained their dissociation curves down to heptanucleotides. Other approaches, e.g., an additional hybridization of short oligonucleotides which continuously extend duplexes formed between the fragment and immobilized oligonucleotides, should considerably increase either the probability of unambiguous reconstruction, or the length of reconstructed sequences, or decrease the size of immobilized oligonucleotides.     

Unrestricted hybridization of oligonucleotides to structure-free DNA

The existence of secondary structure in long single-stranded DNA and RNA is a serious obstacle to the practical use of short oligonucleotide probes (<20-mers). Here, we show that replication of a highly structured DNA in the presence of a unique set of dNTP analogues leads to synthesis of daughter DNA with a significantly reduced level of secondary structure. This replicated DNA, composed of 2-aminoadenine, 2-thiothymine, 7-deazaguanine, and cytosine bases, was readily accessible to tiled 8-mer LNA and 15-mer DNA probes, whereas an unmodified version of the same DNA was inaccessible. Importantly, while the base analogues enhanced probe-target stability, they did not significantly reduce the specificity of base pairing. The availability of structure-free DNA targets should facilitate the use of short oligonucleotide probes and promote development of generic oligonucleotide microarrays.     

Effect of bone decalcification procedures on DNA in situ hybridization and comparative genomic hybridization. EDTA is highly preferable to a routinely used acid decalcifier.

Decalcification is routinely performed for histological studies of bone-containing tissue. Although DNA in situ hybridization (ISH) and comparative genomic hybridization (CGH) have been successfully employed on archival material, little has been reported on the use of these techniques on archival decalcified bony material. In this study we compared the effects of two commonly used decalcifiers, i.e. , one proprietary, acid-based agent (RDO) and one chelating agent (EDTA), in relation to subsequent DNA ISH and CGH to bony tissues (two normal vertebrae, six prostate tumor bone metastases with one sample decalcified by both EDTA and RDO). We found that RDO-decalcified tissue was not suited for DNA ISH in tissue sections with centromere-specific probes, whereas we were able to adequately determine the chromosomal status of EDTA-decalcified material of both control and tumor material. Gel electrophoresis revealed that no DNA could be successfully retrieved from RDO-treated material. Moreover, in contrast to RDO-decalcified tumor material, we detected several chromosomal imbalances in the EDTA-decalcified tumor tissue by CGH analysis. Furthermore, it was possible to determine the DNA ploidy status of EDTA- but not of RDO-decalcified material by DNA flow cytometry. Decalcification of bony samples by EDTA is highly recommended for application in DNA ISH and CGH techniques.     

DNA ploidy and chromosomal imbalances in invasive ductal breast cancer. A comparative study of DNA image cytometry and comparative genomic hybridization (CGH).

Chromosomal imbalances were analyzed in 62 breast cancers with different DNA ploidy by CGH. The results of DNA image cytometry and CGH are consistent with peridiploid and aneuploid cases. The peritetraploid tumors harbored a high number of chromosomal imbalances, as a hint for an unfavorable prognosis. The quantitative analysis of imbalances highlighted the role of different physical constituents of the chromosome, and of chromosomal losses in different DNA ploidy groups. The peritetraploid and aneuploid tumors differed from the peridiploid tumors in losses at 8p and 18q. The peritetraploid cancers exhibited more gains at 8q, the aneuploid tumors more losses at 17p than their peridiploid counterparts. The aneuploid cases differed from the peritetraploid tumors in a higher number of losses at 11q and 14q. Combinations of imbalances provide further insights into the genetic background of DNA ploidy. Hypotheses for the progression from peridiploid to nondiploid breast cancers are given.