This report describes the use of fluorescence in situ hybridization for chromosome classification and detection of chromosome aberrations. Biotin-labeled DNA was hybridized to target chromosomes and subsequently rendered fluorescent by successive treatments with fluorescein-labeled avidin and biotinylated anti-avidin antibody. Human chromosomes in human-hamster hybrid cell lines were intensely and uniformly stained in metaphase spreads and interphase nuclei when human genomic DNA was used as a probe. Interspecies translocations were detected easily at metaphase. The human-specific fluorescence intensity from cell nuclei and chromosomes was proportional to the amount of target human DNA. Human Y chromosomes were fluorescently stained in metaphase and interphase nuclei by using a 0.8-kilobase DNA probe specific for the Y chromosome. Cells from males were 40 times brighter than those from females. Both Y chromosomal domains were visible in most interphase nuclei of XYY amniocytes. Human 28S ribosomal RNA genes on metaphase chromosomes were distinctly stained by using a 1.5-kilobase DNA probe.

https://www.pnas.org/content/pnas/83/9/2934.full.pdf

Cytogenetic analysis using quantitative, high-sensitivity, fluorescence hybridization.

Disclosed are new methods comprising the use of in situ hybridization to detect abnormal nucleic acid sequence copy numbers in one or more genomes wherein repetitive sequences that bind to multiple loci in a reference chromosome spread are either substantially removed and/or their hybridization signals suppressed. The invention termed Comparative Genomic Hybridization (CGH) provides for methods of determining the relative number of copies of nucleic acid sequences in one or more subject genomes or portions thereof (for example, a tumor cell) as a function of the location of those sequences in a reference genome (for example, a normal human genome). The intensity(ies) of the signals from each labeled subject nucleic acid and/or the differences in the ratios between different signals from the labeled subject nucleic acid sequences are compared to determine the relative copy numbers of the nucleic acid sequences in the one or more subject genomes as a function of position along the reference chromosome spread. Amplifications, duplications and/or deletions in the subject genome(s) can be detected. Also provided is a method of determining the absolute copy numbers of substantially all RNA or DNA sequences in subject cell(s) or cell population(s).

https://www.annualreviews.org/doi/pdf/10.1146/annurev.genom.6.080604.162140

Comparative genomic hybridization

The present invention provides methods of determining relative copy number of target nucleic acids and precise mapping of chromosomal abnormalities associated with disease. The methods of the invention use target nucleic acids immobilized on a solid surface, to which a sample comprising two sets of differentially labeled nucleic acids are hybridized. The hybridization of the labeled nucleic acids to the solid surface is then detected using standard techniques.

Comparative fluorescence hybridization to nucleic acid arrays

The c-erbB-2 gene is a v-erbB-related proto-oncogene which is distinct from the gene encoding the epidermal growth factor receptor. By using two independent methods, hybridization of both sorted chromosomes and metaphase spreads with cloned c-erbB-2 DNA, we mapped the c-erbB-2 locus on human chromosome 17 at q21, a specific breakpoint observed in a translocation associated with acute promyelocytic leukemia. Furthermore, we observed amplification and elevated expression of the c-erbB-2 gene in the MKN-7 gastric cancer cell line. These data suggest possible involvement of the c-erbB-2 gene in human cancer.

Localization of a novel v-erbB-related gene, c-erbB-2, on human chromosome 17 and its amplification in a gastric cancer cell line

Publisher Summary This chapter discusses the myc /IgH translocation. It considers the nature of the variant forms of the plasmacytoma and Burkitt translocations. The bcr–abl recombination that creates the Philadelphia chromosome is also described and its role in the development of chronic myeloid leukemia is considered. Three putative oncogenes— pvt-1, bcl-1, and bcE-2 —have been identified at the translocation junctions in lymphoid tumors by the molecular cloning of “aberrant” immunoglobulin gene rearrangements. Several other chromosomal abnormalities specific to particular lymphoid and myeloid neoplasms are assessed in the chapter for the possible involvement of oncogenes. The challenge for the future is not only to delineate the molecular basis for other translocations but also to identify the normal function of the genes involved. The frequent association of abnormalities involving band 11q23 in acute leukemias with monocytic differentiation suggests that they involve the alteration of a gene important in myelomonocytic differentiation. The recent localization of the metallothionein gene cluster to the band q22 of chromosome 16 raises intriguing questions regarding the chromosome abnormalities associated with acute myelomonocytic leukemia (AMML).

Activation of cellular oncogenes in hemopoietic cells by chromosome translocation.

Highly purified fractions of human chromosomes 21 and 22 were isolated from a suspension of metaphase chromosomes stained with ethidium bromide by using a fluorescence-activated cell sorter (FACS II). Two recombinant DNA libraries, representing chromosomes 21 and 22, were constructed by complete digestion of DNA from these fractions with EcoRI and insertion into the vector lambda gtWES lambda B. Twenty clones selected at random from the chromosome 22 library hybridized to EcoRI-digested human DNA, and five of these clones hybridized to single bands identical in size to the phage inserts. These five single-copy sequences and a clone coding for an 8S RNA isolated by screening the chromosome 22 library for expressed sequences were characterized in detail. Hybridization of all six clones to a panel of sorted chromosomes and hybrid cell lines confirmed the assignment of the sequences to chromosome 22. The sequences were localized to regions of chromosome 22 by hybridization to translocated chromosomes sorted from a cell line having a balanced translocation t(17;22)(p13;q11) and to hybrid cell lines containing the various portions of another translocation t(X;22)(q13;q112). Five clones reside on the long arm of chromosome 22 between q112 and pter, while one clone and an 18S rRNA gene isolated from the chromosome 22 library reside pter and g112. The construction of chromosome-specific libraries by this method has the advantage of being direct and applicable to nearly all human chromosomes and will be important in molecular analysis of human genetic diseases.

http://www.pnas.org/content/pnas/79/9/2971.full.pdf

Robert Krumlauf

Papers

Construction and characterization of genomic libraries from specific human chromosomes