A novel technique for measuring variations in DNA copy-number: competitive genomic polymerase chain reaction

Abstract

Background: Changes in genomic copy number occur in many human diseases including cancer. Characterization of these changes is important for both basic understanding and diagnosis of these diseases. Microarrays have recently become the standard technique and are commercially available. However, it is useful to have an affordable technique to complement them.

Results: We describe a novel polymerase chain reaction (PCR)-based technique, termed competitive genomic PCR (CGP). The main characteristic of CGP is that different adaptors are added to the sample and control genomic DNAs after appropriate restriction enzyme digestion. These adaptor-supplemented DNAs are subjected to competitive PCR using an adaptor-primer and a locus-specific primer. The amplified products are then separated according to size differences between the adaptors. CGP eliminates the tedious steps inherent in quantitative PCR and achieves moderate throughput. Assays with different X chromosome numbers showed that it can provide accurate quantification. High-resolution analysis of neuroblastoma cell lines around the MYCN locus revealed novel junctions for amplification, which were not detected by a commercial array.

Conclusion: CGP is a moderate throughput technique for analyzing changes in genomic copy numbers. Because CGP can measure any genomic locus using PCR primers, it is especially useful for detailed analysis of a genomic region of interest.

Figure 1

Outline of the CGP assay. Schematic representation of the CGP assay for quantification using PstI and MboI as restriction enzymes.

Figure 2

A, B. Validation of the CGP assay using Fmix vs. Fmix (A), or Fmix vs. Mmix (B). The horizontal axis displays 132 loci of chromosome X and 60 loci of chromosome 17. The vertical axis represents test/reference log₂ fluorescence ratio of each locus. C, D. Sensitivity of CGP for detecting a change in low-level copy number. CGP was performed to determine the copy number of loci in the chromosome X. Fmix DNA (46, XX) was used as a reference DNA for all reactions. The distribution of test/reference fluorescence ratios for the different test samples of chromosome X DNA (C). Vertical axis, the percentage of DNAs; horizontal axis, test/reference fluorescence ratio. The doublet line, bold line, solid line, dotted line and dashed line indicate the Mmix/Fmix, Fmix/Fmix, 47, XXX/Fmix, 48, XXXX/Fmix and 49, XXXXX/Fmix fluorescence ratios, respectively. The mean relative ratios of autosomal DNAs (dotted line) and chromosome X DNA (solid line) from each experiment versus the number of X chromosomes (D). Mean (± 1 s.d.) fluorescence ratios of chromosome X DNA were as follows; XY versus XX, 0.69 (0.49–0.89); XX versus XX, 1.00 (0.84–1.15); XXX versus XX, 1.37 (1.10–1.64); XXXX versus XX, 1.62 (1.21–2.02); XXXXX versus XX, 2.08 (1.63–2.52). A dotted line for chromosome X and a solid line for autosomal mean fluorescence ratios were fitted using standard regression analysis.

Figure 3

Outline of locus-specific primer design. 'M' and 'P' indicate the MboI and PstI restriction sites, respectively.

Figure 4

Detection of MYCN gene amplification in neuroblastoma cell lines in CGP. A. Confirmation of MYCN amplification in neuroblastoma cell lines by Southern hybridization. B. CGP analysis of neuroblastoma cell lines. DNA copy number profiles for chromosome region 2p25.3-2q14.3 (approximately every 1.3 Mbps) containing the MYCN gene were derived from 96 oligonucleotide primers. All 12 neuroblastoma-derived cell lines were examined, but only the SH-SY5Y, TNB-1, NGP and SK-N-BE lines are displayed. Horizontal axis indicates each locus from 2pter (bp), and vertical axis shows test/reference log₂ fluorescence ratio. The MYCN loci are indicated by black circles. C. CGP high-resolution analysis (approximately every 48 kbps) in neuroblastoma cell lines. DNA copy number profiles for chromosome 2p24.2-2p24.3 containing the MYCN gene were derived from 72 oligonucleotide primers and represented 66 loci. All 12 neuroblastoma-derived cell lines were examined, but only NBL-S, SK-N-DZ, TGW and RTBM1 cell lines are displayed. Horizontal axis indicates each locus from 2pter, and vertical axis shows test/reference log₂ fluorescence ratio. The arrow and black circles denote the MYCN loci. D. Summary of the amplified region surrounding the MYCN gene. The vertical axis indicates the amplified loci between 15.5 Mega (M) bp and 17.5 M bp from 2pter. The black bars denote the DNA amplification sites for each cell line. Regions where amplifications were detected in more than 2 spots of the CGP assay were defined as "amplification sites". The dashed line represents the loci of NAG, DDX1, MYCN and FAM49A.

Figure 5

Detection of MYCN gene amplification in TGW cell line using oligonucelotide arrays. A. DNA copy number changes on chromosome 2. The horizontal axis indicates each locus from 2pter to 2qter, and the vertical axis shows estimated copy number. Gains or losses in DNA copy number are indicated by the blue and red bars, respectively. The brown box depicts the amplified region in chromosome 2 and is expanded in B. B. The region adjacent to the MYCN gene locus highlights chromosomal changes (the brown box region in Fig.5A). The Integrated Genome Browser (IGB) was used for visualization of the data. The horizontal axis indicates chromosome locus, and the vertical axis shows estimated copy number. The two red lines indicate 15.5 M bp and 17.5 M bp, respectively, as in Fig. 4D.