Fluorescence in situ hybridization with high-complexity repeat-free oligonucleotide probes generated by massively parallel synthesis

Abstract

The ability to visualize specific DNA sequences, on chromosomes and in nuclei, by fluorescence in situ hybridization (FISH) is fundamental to many aspects of genetics, genomics and cell biology. Probe selection is currently limited by the availability of DNA clones or the appropriate pool of DNA sequences for PCR amplification. Here, we show that liquid-phase probe pools from sequence capture technology can be adapted to generate fluorescently labelled pools of oligonucleotides that are very effective as repeat-free FISH probes in mammalian cells. As well as detection of small (15 kb) and larger (100 kb) specific loci in both cultured cells and tissue sections, we show that complex oligonucleotide pools can be used as probes to visualize features of nuclear organization. Using this approach, we dramatically reveal the disposition of exons around the outside of a chromosome territory core and away from the nuclear periphery.

Fig. 1

Detection of specific loci with small sequence capture pools. a Metaphase chromosomes from mouse ES cells hybridized with 6FAM (green) 130 kb Hoxd sequence capture probe pool. DNA was stained with DAPI. Scale bar = 5 μm. b Mouse metaphase chromosomes hybridized with TexasRed 15 kb sequence capture probe for Hoxd1 (Table 1). Scale bar = 5 μm. c Efficiency with which probe pools of decreasing genomic target size detect loci on the chromatids of metaphase chromosomes (n > 160). d As in (a) but hybridization to nuclei in a mouse embryo section. Scale bar = 3 μm

Fig. 2

Detection of specific loci and whole chromosomes with sequence capture pools. a Metaphase chromosome spread and interphase nucleus from mouse ES cells hybridized with TexasRed MMU2 exome sequence-capture probe pool without the addition of any mouse CotI DNA for repeat suppression. DNA was stained with DAPI. Scale bar = 5 μm. b Metaphase chromosomes from mouse ES cells hybridized with TexasRed MMU2 exome sequence capture probe pool and FITC-labelled (green) standard MMU2 chromosome paint (Cambio). Scale bar = 5 μm. Grey-scale images of the red exome paint signal and the green chromosome paint are shown below. c Mouse ES cell nucleus (top) and metaphase chromosomes (bottom) co-hybridized with TexasRed MMU2 exome and 6FAM (green) 130 kb Hoxd sequence capture pools. Scale bar = 3 μm. d As in (c) but hybridization to mouse embryo section. Scale bar = 3 μm

Fig. 3

Analysing the nuclear organization of chromosomes. a Left, interphase nucleus from mouse ES cells hybridized with TexasRed MMU2 exome sequence capture probe pool and FITC-labelled (green) standard MMU2 chromosome paint (Cambio). Scale bar = 5 μm. Grey-scale images of the red exome paint signal and the green chromosome paint are shown to the right. b Box plots showing the proportion of nuclear area covered by hybridization signal from the standard MMU2 chromosome paint (green) and the MMU2 exome oligonucleotide pool (red). Boxed area shows the median and interquartile range; asterisks indicate outliers. n > 198 chromosome territories. c Erosion analysis to assess the distributions of hybridization signals from TexasRed MMU2 exome sequence capture probe pool (red) and FITC-labelled MMU2 chromosome paint (green), relative to that of DNA (DAPI), across five concentric shells from the edge (shell 1) to the centre (shell 5) of the nucleus. Graph shows mean (+/– SEM) percent hybridization signal in a particular erosion shell, normalized to the percentage of DAPI in that shell, averaged from 68 ES cell nuclei