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Multicolor FISHs for simultaneous detection of genes and DNA segments on human chromosomes

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Abstract

We have developed a convenient multicolor fluorescent in situ hybridization (FISH) (five-, four-, three-, and two-color FISHs) for detecting specific genes/DNA segments on the human chromosomes. As a foundation of multicolor FISH, we first isolated 80 bacterial artificial chromosome (BAC) probes that specifically detect the peri-centromeres (peri-CEN) and subtelomeres (subTEL) of 24 different human chromosomes (nos. 1~22, X, and Y) by screening our homemade BAC library (Keio BAC library) consisting of 200,000 clones. Five-color FISH was performed using human DNA segments specific for peri-CEN or subTEL, which were labeled with five different fluorescent dyes [7-diethylaminocoumarin (DEAC): blue, fluorescein isothiocyanate (FITC): green, rhodamine 6G (R6G): yellow, TexRed: red, and cyanine5 (Cy5): purple]. To observe FISH signals under a fluorescence microscope, five optic filters were carefully chosen to avoid overlapping fluorescence emission. Five-color FISH and four-color FISH enabled us to accurately examine the numerical anomaly of human chromosomes. Three-color FISH using two specific BAC clones, that distinguish 5′ half of oncogene epidermal growth factor receptor (EGFR) from its 3′ half, revealed the amplification and truncation of EGFR in EGFR-overproducing cancer cells. Moreover, two-color FISH readily detected a fusion gene in leukemia cells such as breakpoint cluster region (BCR)/Abelson murine leukemia viral oncogene homologue (ABL) on the Philadelphia (Ph′) chromosome with interchromosomal translocation. Some other successful cases such as trisomy 21 of Down syndrome are presented. Potential applications of multicolor FISH will be discussed.

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Abbreviations

ABL:

Abelson murine leukemia viral oncogene homologue

BAC:

Bacterial artificial chromosome

BCR:

Breakpoint cluster region, peri-CEN (peri-centromere)

CML:

Chronic myeloid leukemia

Dyrk1A:

Dual specificity tyrosine-phosphorylation-regulated kinase 1A

EGF:

Epidermal growth factor

EGFR:

Epidermal growth factor receptor

ES:

Embryonic stem

FISH:

Fluorescent in situ hybridization

iPS:

Induced pluripotent stem

PCR:

Polymerase chain reaction

Ph′:

Philadelphia

TEL:

Telomere

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Acknowledgments

The authors thank Dr. S. Asakawa for his initial contribution to the construction and characterization of Keio BAC library. The authors also thank Drs. S. Minoshima, J. Kudoh, A. Takayanagi, and many postdocs/graduate students for their contribution in exploring the new use of BAC clones for molecular studies of human genome.

Conflict of interest

The authors (MM, SA, YS, and NS) declare that they have no conflict of interest. This article does not contain any studies with human or animal subjects performed by any of the authors.

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Authors and Affiliations

  1. Keio University, Tsukuba, Ibaraki, Japan

    Nobuyoshi Shimizu, Masahiko Maekawa, Satoko Asai & Yoshiko Shimizu

Authors
  1. Nobuyoshi Shimizu
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  2. Masahiko Maekawa
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  3. Satoko Asai
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  4. Yoshiko Shimizu
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Corresponding author

Correspondence to Nobuyoshi Shimizu.

Additional information

Responsible Editor: Beth A. Sullivan

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Figure S1

Chromosomal location of BAC clones for peri-centromeres and sub-telomeres to use as a foundation of multi-color FISH. BAC clones that distinguish peri-centromeres (peri-CENs) and sub-telomeres (subTELs) in 24 different human chromosomes (#1~#22, X and Y) were chosen to use as foundation of multi-color FISH. (TIFF 1520 kb)

High Resolution (GIF 156 kb)

Figure S2

Screening of BAC clones from Keio BAC library. Screening was carried out by rapid 2-step PCR method using Keio-BAC library consisting of 200,000 clones (a). Ca. 300 96-well micro-titer plates are stocked in a deep freezer (b). Human DNA segments were cloned into B AC vectors and grown in E. coli (c). (TIFF 1520 kb)

High Resolution (GIF 250 kb)

Figure S3

BAC contigs and gene clusters in the region of chromosome 22q11.2. Significant numbers of BAC clones were used for human genome project and aligned on particular chromosomes, showing aligned BAC contigs, clusters of immunoglobulin light chain genes in the region of chromosome 22q11.2. Many other known genes (red bars), new genes (green bars) and pseudogenes (green bars) as well as DNA segments (many of black bars) are indicated. (TIFF 1520 kb)

High Resolution (GIF 230 kb)

Figure S4

BAC-ends sequencing and chromosomal mapping. Both ends of more than 20,000 clones were sequenced (a) and they were mapped on particular human chromosomes based on the genome sequence data (b). (TIFF 1520 kb)

High Resolution (GIF 299 kb)

Figure S5

The best combination of fluorescent dyes (up to 5) and optic filters for 5-color-FISH. The best combination of fluorescent dyes and filters was found to be DEAC:SpAqua-C, FITC:SpGr-B, R6G:SpGold-B, TexRed:SpRed-B, Cy5:CY5-4040B (Semrock, Inc.). Labeling was carried out by nick-translation and subjected to conventional FISH procedure. Since Cy5 is infrared light and invisible by naked eye, the result was drawn with artificial color (purple). Olympus BX51with CCD camera system DP71 was used with selected optic filters. (TIFF 1520 kb)

High Resolution (GIF 118 kb)

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Shimizu, N., Maekawa, M., Asai, S. et al. Multicolor FISHs for simultaneous detection of genes and DNA segments on human chromosomes. Chromosome Res 23, 649–662 (2015). https://doi.org/10.1007/s10577-015-9473-9

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  • DOI: https://doi.org/10.1007/s10577-015-9473-9

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