Comparative genomic hybridization, loss of heterozygosity, and DNA sequence analysis of single cells

Abstract

A PCR strategy is described for global amplification of DNA from a single eukaryotic cell that enables the comprehensive analysis of the whole genome. By comparative genomic hybridization, not only gross DNA copy number variations, such as monosomic X and trisomic 21 in single male cells and cells from Down's syndrome patients, respectively, but multiple deletions and amplifications characteristic for human tumor cells are reliably retrieved. As a model of heterogeneous cell populations exposed to selective pressure, we have studied single micrometastatic cells isolated from bone marrow of cancer patients. The observed congruent pattern of comparative genomic hybridization data, loss of heterozygosity, and mutations as detected by sequencing attests to the technique's fidelity and demonstrates its usefulness for assessing clonal evolution of genetic variants in complex populations.

Figure 1

Isolation of single fluorescent cells from bone marrow by micromanipulation and preparation of DNA after proteinase K (PK) treatment. After PK inactivation, the double-stranded DNA was digested with MseI, leaving a TA overhang for adapter annealing and subsequent ligation. After primary amplification, 1/100 of the PCR products was reamplified in the presence of bio-UTP, and 2 μg were used for hybridization.

Figure 2

CGH profile of a single peripheral blood leukocyte of a male patient with Down’s syndrome. To be considered significant, deviations from the central line had to cross the green (chromosomal gain) or red (chromosomal loss) punctate line. The gray shaded horizontal bars indicate regions excluded from analysis because of the prevalence of heterochromatic DNA. The chromosome 21 (except of the blocked heterochromatic region) was found to be amplified entirely whereas all other chromosomes showed normal profiles.

Figure 3

Comparison of the CGH profiles of a “conventional” CGH experiment using 1 μg of nick-translated DNA prepared from the MCF-7 cell line and from four single cells of the same cell line after PCR amplification. Of all profiles, the results of chromosome 2 are shown. The green bar on the right side of the chromosome 2 symbol of single cell 1 indicates the partial chromosomal gain only detected in this cell but not in any other single cell or the pooled DNA of the cell line.

Figure 4

Two-color interphase-FISH on MCF-7 cells with YAC clones specific for 2p25 (red) and 2q31 (green). In one cell (upper left corner), three signals can be identified for the 2p region, but only two can be identified for 2q. The other cells show two signals for both the 2p and the 2q probes.

Figure 5

Isolation of a single disseminated tumor cell from bone marrow of a breast cancer patient. (A) Nucleated cell suspension containing one immunofluorescent cytokeratin-positive cell. (B) Exclusion of visible light, demonstrating the bright cytoplasmic staining and the negative background of surrounding cells. The fluorescent cell then was aspirated by a glass capillary (C) and was transferred to a fresh slide. No cells other than the fluorescent cell were transferred because the whole visual field did not contain any other cell. From there, the cell was taken to the amplification tube.

Figure 6

CGH profile of four single tumor cells (T#1–T#4) and one normal cell of a patient with CUP syndrome. Chromosomes 2, 5, 7, and 8 were chosen for comparison of identical and divergent findings. The losses on chromosomes 2, 5, and 8p were common for all tumor cells as was the gain of 8q whereas the loss of chromosome 7 was only seen for T#2. As in the other profiles, chromosomal losses are marked by red vertical bars at the left side of the chromosome symbol, and gains are marked by a green bar on the right side.

Figure 7

(A) LOH analysis using the dinucleotide repeat polymorphism at D5S346 locus linked to the APC gene (Upper) and PCR–restriction fragment length polymorphism of a Alw21I site within exon 15 of the APC gene (Lower). In the first experiment, all four tumor cells demonstrated loss of one allele whereas the single control cells contained two alleles, except for control cell 2. In the APC PCR–restriction fragment length polymorphism experiment, all but one control cell (3) had two alleles whereas the tumor cells only contained the uncut fragment. The fragment of tumor cell 4 was not amplified. −, negative control; M10, 10-bp ladder; M50, 50-bp ladder; M, marker for agarose gel electrophoresis. (B) Sequence of the mutation found in codon 215. The single control cells contained the wild-type sequence with an A at nucleotide 643 (upper two sequences) whereas the tumor cells were mutated at this position showing an A → G mutation (lower two sequences). The sequences of four of the eight cells are shown.