Use of fluorescent sequence-specific polyamides to discriminate human chromosomes by microscopy and flow cytometry

Abstract

In this paper, we demonstrate the use of synthetic polyamide probes to fluorescently label heterochromatic regions on human chromosomes for discrimination in cytogenetic preparations and by flow cytometry. Polyamides bind to the minor groove of DNA in a sequence-specific manner. Unlike conventional sequence-specific DNA or RNA probes, polyamides can recognize their target sequence without the need to subject chromosomes to harsh denaturing conditions. For this study, we designed and synthesized a polyamide to target the TTCCA-motif repeated in the heterochromatic regions of chromosome 9, Y and 1. We demonstrate that the fluorescently labeled polyamide binds to its target sequence in both conventional cytogenetic preparations of metaphase chromosomes and suspended chromosomes without denaturation. Chromosomes 9 and Y can be discriminated and purified by flow sorting on the basis of polyamide binding and Hoechst 33258 staining. We generate chromosome 9- and Y-specific 'paints' from the sorted fractions. We demonstrate the utility of this technology by characterizing the sequence of an olfactory receptor gene that is duplicated on multiple chromosomes. By separating chromosome 9 from chromosomes 10-12 on the basis of polyamide fluorescence, we determine and differentiate the haplotypes of the highly similar copies of this gene on chromosomes 9 and 11.

Figure 1

Comparison of the binding of DNA oligomer and polyamides designed to recognize TTCCA repeats in metaphase chromosomes from a normal male. (A) Conventional FISH of the 20-nt (TTCCA)₄ oligomer to denatured chromosome preparations. Hybridization sites are labeled with avidin–fluorescein (pseudocolored red), and chromosomes are counterstained with DAPI (gray). (B and D) Binding of the polyamides S-PA-F and L-PA-F to non-denatured chromosomes, respectively. The polyamides are responsible for all visible signals (intensity displayed as gray scale). (C and E) Relative intensity profile plots for chromosomes 9, Y and 1 from (B) and (D), respectively. The plotted intensities represent the sum of signal intensities in a seven-pixel-wide swath running from the p-terminus to q-terminus for each chromosome.

Figure 2

Molecular recognition of the 5′-TTCCA-3′ target sequence by the polyamides S-PA-F and L-PA-F. (A) Binding model for the complex formed between S-PA-F and 5′-ATTCCA-3′. (B) Binding model for the complex formed between L-PA-F and 5′-ATTCCATTCCA-3′. (A and B) Filled circles, Im; open circles, Py; diamond, β; (+), DA. Curvature of the polyamide chain to form a hairpin is achieved with a γ linker, and fluorescence is conferred by a fluorescein derivative (AEEA-C6-F) (represented by the F in a gray circle). (C) Hydrogen bonding patterns of nucleotide base pairs with polyamide subunits. Dashed lines represent hydrogen bonds.

Figure 3

Bivariate flow analyses of chromosomes treated with HO and the polyamide S-PA-F (A) or CA (B). Numbers represent the chromosome(s) responsible for the peaks in the flow karyotypes, with lines between the two plots drawn to indicate the positions of chromosomes 1, 9 and Y on the HO scales. Blue- and red-boxed areas in (A) denote sorting windows used to sort chromosomes analyzed further in Figures 4 and 5. Each plot represents measurements of ∼200 000 chromosomes.

Figure 4

Chromosome paints generated from chromosomes sorted on the basis of the intensity of bound S-PA-F polyamide and HO. The chromosomes used to produce the paints in (A)–(D) correspond to the sort windows a–d in Figure 3A. In each case, 2000 chromosomes were sorted, amplified and biotinylated by DOP-PCR, hybridized to metaphase chromosomes and detected with fluorescein–avidin. The fluorescein-bright chromosomes in sort window a yield a chromosome 9-specific paint (A); the fluorescein-dim chromosomes with similar HO fluorescence intensities (sort window b) paint chromosomes 10, 11 and 12 (B). The fluorescein-bright chromosomes in sort window c yield a chromosome Y-specific paint (the long arm is unlabeled due to the suppression of the hybridization of highly repetitive sequences in the paint probe) (C); the fluorescein-dim chromosomes with a similar HO fluorescence intensity (sort window d) paint chromosome 18 (D). All metaphases were counterstained with DAPI.

Figure 5

Sequence analysis of a duplicated block encompassing the OR-A olfactory receptor gene derived from chromosomes 9 and 11 of the CGM1 cell line. Previous FISH analyses had demonstrated the presence of a copy of this block on one homolog of chromosome 9 and both homologs of chromosome 11 in this individual in addition to six other sites (both homologs of 3, 15 and 19). Sorted chromosomes were subjected to PCR to amplify a region of OR-A corresponding to bases 16 147–17 307 in GenBank accession no. L78442. The PCR product was used as template for the sequencing reaction after removal of PCR primers and nucleotides. (Top) The sequence generated when chromosomes 9–12 were sorted together on the basis of HO and CA staining. The arrow indicates the site of a single-nucleotide variation among the copies of OR-A found on these chromosomes. Two peaks, corresponding to a thymine (T) (red) and guanine (G) (black), are observed at nt 17 015. (Middle) Sequence of the block on chromosome 9, obtained by PCR amplification of chromosomes sorted on the basis of S-PA-F polyamide and HO fluorescence intensities (sort window a; Fig. 3A). A single peak corresponding to thymine is observed at nt 17 015. (Bottom) Sequence of the block on chromosome 11, obtained by PCR amplification of chromosomes 10–12 sorted on the basis of polyamide and HO fluorescence (sort window b; Fig. 3A). The arrow denotes a single peak, corresponding to a guanine at this position. Other nucleotides represented are cytosine (C) (blue) and adenine (A) (green).