We present a robust, standardized protocol for the analysis of genome copy-number alterations in DNA derived from as few as 1000 microdissected cells from histology specimens. The
29 polymerase-based MDA generates high-molecular DNA replication products in high yields, and is suitable when processing large numbers of samples. Some representational distortion occurs, which is likely to result from variability in priming density and processing by the
29 polymerase of repetitive and polymorphic sequences. Similar systematic changes in copy-number structure were reported in other array CGH studies using
29-amplified DNA (15
). Some amplification biases can be partially compensated for by using test and reference samples amplified under the same conditions (10
). We compared the results of co-hybridization of our test sample with amplified and unamplified reference DNA samples, and surprisingly found similar ratio distributions. Since previous studies used either a different strand displacement polymerase (Bst) and cDNA microarrays (15
), or a PCR-based amplification method (10
), these results may not be directly comparable to ours. Moreover, Lage et al
. also successfully used an unamplified reference sample in an evaluation experiment on human BAC arrays (15
). Taken the fact that the use of an unamplified reference is simpler and less expensive for high-throughput analyses of many samples, we opted for an unamplified reference. As we show, the observed over- and under-representations of specific genomic regions were reproducible and as such can be handled with adequate statistical tools. By taking individual clone-specific effects into account in our statistical analyses, they were dealt with in an effective way without excluding clones based on an arbitrary threshold. This approach allowed us to analyze the whole genome, including the Y chromosome, which is routinely excluded entirely due to variable hybridization results, even in classical array CGH (6
Using the X chromosome as a model, we show that our amplified array CGH method detects monosomy and trisomy of the whole chromosomes using the linear regression model. It was even possible to detect a loss of one copy in 50% of the cells, while a similar sensitivity was not reached for a single copy gain. The observed underestimation of the magnitude of copy-number changes has been previously reported in array CGH studies, and most likely results from incomplete suppression of repetitive sequences or errors in background subtraction (6
). However, for all the analyzed X chromosome dosage levels, the relationship between the estimated and the expected ratios were linear, indicating that the amplitude of compression is constant over these levels. As the copy number departed farther from the genome average, the variance of the ratios measured for the X and Y chromosome clones increased. These ratio variations were reproducible, suggesting that the sequence characteristics of individual clones, possibly differing amounts of sequence shared between the X and Y chromosomes, play a role (6
). To establish a limit of accuracy for small regions of gains or deletions, we applied amplified array CGH to a sample known to harbor a trisomic region spanning five BAC clones (5 Mb) on chromosome 20. This aberration was readily detected using a simple, nonparametric Smith–Waterman dynamic algorithm. The lack of assumptions in this method makes its use very convenient. For our purpose, it demonstrated good sensitivity and specificity indicating that the resolution of our amplified array CGH method is at least 5 Mb. Both statistical models have a very low false discovery rate, when log2
ratio thresholds compared to the intercept of −0.35 (0.78 in linear scale) and 0.35 (1.27 in linear scale), representing a biologically meaningful gain or loss, are applied in addition to a significant p
-value. Our data also show the importance of using a pilot study, with normal and control samples carrying known copy-number changes. The generated data can be subsequently used for calibration and for sensitivity determination in the analytical approach.
Applying the novel LCM-array CGH protocol to the analysis of normal colon mucosa and colonic adenomatous polyps from FAP patients provided proof of principle. A subchromosomal 5q deletion was detected by LCM-array CGH analysis and confirmed by an independent PCR-based method. Although we have not tested the minimum amount of input DNA for the MDA reaction, the recommended minimal amount is 1 ng, representing approximately 300–500 human genomic equivalents.
As shown here, cryo-preserved tissues provide excellent starting material for
29 amplification. However, the amplification efficiency is reduced proportionally to a decrease in molecular weight of the starting material, which is problematic for amplification of formalin-fixed archival DNA (15
). Recently, several other methods for whole-genome amplification in combination with array CGH were described. Balanced-PCR amplification (36
) employs digestion and ligation of a target and control genome with distinct linkers, which are mixed and amplified in a single PCR, thereby avoiding biases associated with PCR saturation and impurities. This procedure showed equivalent performance compared to MDA on cDNA microarrays using intact genomic DNA, but overcomes problems associated with modest DNA degradation in formalin-fixed paraffin-embedded tissues (36
). Guillaud-Bataille et al
. present an optimized LM-PCR protocol using 1 ng of starting DNA and BAC arrays. This approach preserves the initial ratios observed with BAC array CGH, allowing the reliable detection of one-copy-level variations among the amplified material (10
). Although this method has not yet been applied to microdissected samples, the results on cell line DNA are very promising.
cDNA arrays typically consisting of several tens of thousands of features are used for array CGH experiments because of their more common availability and higher resolution (15
). Compared to BAC arrays, however, the signal-to-noise ratio is lower and signals of two to five neighboring clones are averaged to improve signal reproducibility. To reach a similar resolution using BAC arrays, around 10
000 BACs would be necessary, resulting in a tiling array for the human genome (39
). Since the size of the BACs, 150–200 kb, ultimately obscures higher resolution, microarrays containing 25mer (38
) or 60–70mer (42
) oligonucleotide probes are currently being explored for measuring DNA copy-number changes. The commercially available synthetic 25mer high-density oligonucleotide arrays, which were originally designed to detect single-nucleotide polymorphisms (SNPs) (45
), have the advantage of giving genotyping data in conjunction with copy-number analysis (38
). Overall, this platform exhibited more variability than BAC array-based CGH, and while high-level amplifications and homozygous deletions were reliably reported, changes resulting in loss or gain of a single copy were often missed in unamplified tumor cell line DNA (38
). It is anticipated that as SNP density increases, resolution and the ability to assess subtle copy-number changes will increase. Importantly, in a small-scale study, Wong et al
. used SNP arrays in combination with
29-amplified DNA from two tumor biopsies, and showed good concordance between amplified and unamplified DNA for a high-level amplification and deletion (40
). This study shows the feasibility of combining MDA whole-genome amplification with sensitive copy-number analysis at high resolution.
In summary, we show that the strand displacement polymerase
29 reproducibly amplifies starting amounts of genomic DNA as low as 2 ng or 1000 laser-capture microdissected cells, resulting in the reliable detection of single copy variations on BAC array CGH. This method allows the detection of specific genetic alterations in small neoplastic lesions, including tumor biopsies obtained by non-surgical sampling methods like endoscopies, and in clinically relevant subpopulations of tumor cells, e.g. invading fronts of tumors. The significance of the capability to detect single-copy alterations in tissue samples consisting of only a few thousand cells, lies in the greatly expanded potential for discovery of novel genetic alterations limited to small clonal patches in tumors, or present in small preneoplastic lesions, as demonstrated here by the detection of the subchromosomal 5q deletion in a FAP-derived adenomatous polyp by combined LCM-array CGH analysis. These features will facilitate the identification of novel oncogenes or tumor suppressors mapping to regions of gene gain or loss (6
). An additional application in medical genetics is the detection of copy-number changes in DNA derived from buccal swaps for diagnosis of congenital chromosomal abnormalities, such as microdeletions and duplications, and unbalanced chromosomal translocations.