Gel electrophoresis analysis of amplified DNA. (A) Control reactions were incubated for 5 h in a 30-μL volume containing no DNA, or 7.5 ng of human DNA. Samples representing 5% of the reaction were denatured in alkaline buffer and analyzed on a 0.5% alkaline agarose gel, and stained with SYBR-green II (Molecular Probes). Lanes labeled M contained phage λ DNA digested with restriction endonuclease HindIII. Reactions catalyzed by φ29 polymerase were incubated with (+) or without (−) input of denatured human DNA. Random heptamers contained standard (−) DNA, or were modified by the addition of two nitroindole groups (+) at the 5′ end. (B) Time-course reactions for φ29 and Bst DNA polymerases. Reactions were performed using nitroindole-modified primers. Every hour (from 1–5 h), 1.5 μL was removed, denatured in alkaline buffer, and analyzed in 0.5% alkaline agarose gel. Lanes labeled C correspond to control samples incubated for 5 h without input DNA. The lane labeled G corresponds to a gel load of genomic DNA equivalent to 100× the original DNA input of the amplification reactions. (C) Plots under gel images display the time course (fold amplification vs. time) of both polymerase reactions, generated by quantification of DNA yield with the PicoGreen Quantitation Kit. Background fluorescence at time 0 was subtracted for all time points. Each point represents the mean (±1 SD) of four independent analyses.

Evaluation of DNA amplification bias using array–CGH on human cDNA microarrays. (A) To assess the extent of dynamic range compression, three microarray–CGH experiments were performed in duplicate using genomic female DNA (46, XX) versus genomic male DNA, or DNA from cell lines containing 3 X-chromosomes (47, XXX) and 5 X-chromosomes (49, XXXXX) with a normal number of autosomes against genomic male DNA. Autosomal genes located in Chromosomes 1 and 2 are compared with genes located in Chromosome X for the same set of experiments. Ratio values correspond to the average of two independent experiments, and are displayed in a log₂ scale. Averaging the log₂ ratios for the X-linked probes in the three different experiments gives values of 0.234 ± 0.143 (1.176 in linear scale, average of 112 probes), 0.423 ± 0.220 (1.341 in linear scale, average of 113 probes), and 0.681 ± 0.290 (1.603 in linear scale, average of 115 probes) for the 2X, 3X, and 5X experiments, respectively. Dynamically compressed ratios can be converted to actual ratios by fitting log₂ values to a power-law mathematical formula as described by Yuen et al. (2002). (B) Comparison of DNA polymerase-induced representational distortion using human DNA samples. Normal human DNA was amplified with either φ29 or Bst DNA polymerase, labeled with Cy3, and hybridized against similarly amplified human DNA labeled with Cy5. Plots for Chromosomes 1 and 2 are shown in the same scale as the plot in A. (C) Confidence limits for array–CGH analysis of human DNA. Plots correspond to unamplified human female versus male DNAs and whole genome Bst-amplified human female versus Bst-amplified male DNAs. Average log₂ fluorescence ratios for replicate spots are ordered according to the chromosome number and the position in the chromosome. Ratio values for X-linked genes show a similar distribution to that observed for the 2X dosage in A. Confidence limits (horizontal dashed lines) for 99.9% of data for autosomal genes are between −0.262 and 0.262 (0.833 and 1.199 when expressed as linear ratios) for the unamplified experiment. The same confidence bounds calculated for the unamplified experiment are replicated in the plot of ratios generated by microarray analysis of amplified DNA.

Enlarged representation of array–CGH data for Chromosomes 17 and 20. Chromosomes 17 and 20 are known to contain notorious amplifications in the breast cancer cell line BT474. Chromosome 17 is represented in the microarray by 221 cDNAs, and Chromosome 20 by 99 cDNAs. Note that the profiles for Chromosomes 17 and 20 are mostly conserved for the whole genome-amplified samples from different inputs of DNA (equivalent to 500 and 1000 cells) compared with one of the unamplified experiments. The number of altered genes and percentage of concordance of the amplified experiments to the unamplified control are presented in the tables below the plots. The values in diagonal show the total number of altered genes in every experiment for Chromosomes 17 and 20 separately. The first row of each table contains the number of altered genes in common for every amplified experiment compared with the unamplified control. Likewise, the first column of each table gives the percentage of concordance for altered genes found in common between each amplified experiment and the total number of altered genes in the unamplified control. Nonparametric bounds are −0.642 and 0.642 for the unamplified experiment, −0.546 and 0.546 for the experiment using amplified DNA from 1000 cells, and −0.574 and 0.573 for the experiment using amplified DNA from 500 cells.

Analysis of breast cancer cell line MCF7 using BAC clones located in Chromosome 20. The graph on the top shows the copy-number variation for the array–CGH experiment corresponding to the hybridization of genomic DNA from the cell line MCF7 against female DNA. The graph in the middle shows the equivalent experiment performed with isothermal whole genome-amplified DNA from MCF7 against female DNA. Regions of copy number increase are remarkably conserved for the isothermal whole genome-amplified DNA from MCF7. The lower left scatter plot shows the high correlation obtained for the ratios of the three loci showing the largest amplification. The lower right scatter plot shows the correlation coefficient for all the ratios in both experiments.

Schematic representation of the hyperbranched strand-displacement amplification reaction. Single-stranded genomic DNA serves as the target for multiple random priming events. Growing strands are propagated by a DNA polymerase with strand-displacement activity. The 5′ end of each strand is displaced by another upstream strand, growing in the same direction. Displaced strands, which are single-stranded, are now targeted by new random priming events, and these new strands are elongated in the opposite direction. As the reaction proceeds, the hyperbranched network expands dramatically, generating thousands or even millions of copies of the original DNA.

Evaluation of amplification bias using array–CGH on yeast cDNA microarrays. Microarrays contained 6135 unique yeast ORFs. Fluorescence ratios were measured and plotted against the order of the genes in the genome, starting from Chromosome I to Chromosome XVI. (Upper left panel) Analysis of a microarray hybridized with the same DNA, labeled with Cy3 and Cy5. (Upper right panel) DNA from the yeast KO strain was amplified using φ29 DNA polymerase, labeled with Cy3, and hybridized against unamplified (Cy5) DNA from the same strain. (Lower left panel) DNA from the yeast KO strain was amplified using Bst DNA polymerase, labeled with Cy3, and hybridized against unamplified (Cy5) DNA from the same strain. (Center left panel) DNA from the yeast KO strain was amplified using φ29 DNA polymerase for only 2 h, labeled with Cy3, and hybridized against unamplified (Cy5) DNA from the same strain. (Center right panel) Equivalent experiment using Bst DNA polymerase. (Lower right panel) DNAs from the two different yeast strains were amplified to the same extent using Bst and hybridized together. The three genes known to be deleted appear as outlier data points indicated by arrows. The other two outlier data points, near genes GIN4 and CLA4, have abnormally low area values of 48 and 21 according to the Spot analysis software, compared with the average of 255 for all the spots in the array. This abnormality could be produced by a fluorescent speckle over the spot, resulting in unreliable ratios.

Array–CGH of cancer cell line BT474. (A) DNA equivalent to 300,000 cells from breast cancer cell line BT474 was cohybridized with a similar amount of normal human female DNA from peripheral blood lymphocytes to a cDNA microarray containing replicate probes for 4600 genes. BT474 to female fluorescence log₂ ratio profiles were ordered by chromosomes, and into each chromosome, according to the position as assessed by SOURCE (http://genome-www4.stanford.edu/cgi-bin/SMD/source/sourceSearch). Ratio values were connected by lines. A log₂ ratio near 0 indicates an equal copy-number representation of that gene in both genomes. Dashed lines at −0.642 and 0.642 represent nonparametric bounds for log₂ ratios as described in Methods. (B) DNA from 1000 cells, obtained from the BT474 cell line, or DNA from peripheral lymphocytes of a normal human female was amplified using large fragment Bst DNA polymerase and cohybridized to the microarray in the same conditions as before. Nonparametric bounds for this experiment are denoted with dashed lines at −0.546 and 0.546. Arrows indicate gene-dosage alterations observed in both the unamplified and the whole genome-amplified experiments except for shaded areas in Chromosomes 17 and 20 in both figures. These chromosomes are very rich in altered genes, and can be observed in more detail in Figure 6.

 Repeatability of observed log₂ ratios for the set of 15 genes that were identified as significantly amplified in all BT474 experiments. For each experiment, genes were selected using nonparametric methods that flagged log₂ ratios beyond the third quartile plus 2.5 times the Interquartile Range of the observed distribution. (Unamplified BT474) Corresponds to the comparison of unamplified BT474 DNA versus unamplified female DNA. (Amplified from 1000 cells) and (Amplified from 500 cells) correspond to the comparison of amplified DNA from inputs equivalent to 1000 and 500 BT474 cells, respectively, against amplified normal female DNA from similar inputs. On the right side of the plot are shown the actual average ratios and standard deviations for X-chromosome copy-number variations corresponding to ratios of 2:1, 3:1, and 5:1. Using the average value for all four ERBB2 ratios (1.791 in log₂ scale) and the calibration curve derived from the average ratios for 2X, 3X, and 5X (0.234, 0.423, and 0.681, respectively, in log₂ scale), we calculated a hypothetical ratio of 12.5 for this gene. This ratio value is in agreement with amplification values determined by other methods (Lucito et al. 1998), that reported copy number increases in the range of 10–15 for ERBB2 in the breast cancer cell line BT474. Seven of the amplified genes are located in the chromosomal arm 17q (among them PIP2KB, MNL64, ERBB2, and HOXB5), and six probes are located in the chromosomal arm 20q (among them RAE1 and PCK1).