Optimizing PCR Assays for DNA Based Cancer Diagnostics
Somatically acquired DNA rearrangements are characteristic of many cancers. The use of these mutations as diagnostic markers is challenging, because tumor cells are frequently admixed with normal cells, particularly in early stage tumor samples, and thus the samples contain a high background of normal DNA. Detection is further confounded by the fact that the rearrangement boundaries are not conserved across individuals, and might vary over hundreds of kilobases. Here, we present an algorithm for designing PCR primers and oligonucleotide probes to assay for these variant rearrangements. Specifically, the primers and probes tile the entire genomic region surrounding a rearrangement, so as to amplify the mutant DNA over a wide range of possible breakpoints and robustly assay for the amplified signal on an array. Our solution involves the design of a complex combinatorial optimization problem, and also includes a novel alternating multiplexing strategy that makes efficient detection possible. Simulations show that we can achieve near-optimal detection in many different cases, even when the regions are highly non-symmetric. Additionally, we prove that the suggested multiplexing strategy is optimal in breakpoint detection.
We applied our technique to create a custom design to assay for genomic lesions in several cancer cell-lines associated with a disruption in the CDKN2A locus. The CDKN2A deletion has highly variable boundaries across many cancers. We successfully detect the breakpoint in all cell-lines, even when the region has undergone multiple rearrangements. These results point to the development of a successful protocol for early diagnosis and monitoring of cancer.
KeywordsSimulated Annealing Multiplex Reaction CDKN2A Locus Multiple Rearrangement CDKN2A Deletion
Unable to display preview. Download preview PDF.
- 1.Campbell, P., Stephens, P., Pleasance, E., O’Meara, S., Li, H., Santarius, T., Stebbings, L., Leroy, C., Edkins, S., Hardy, C., et al.: Identification of somatically acquired rearrangements in cancer using genome-wide massively parallel paired-end sequencing. Nature Genetics 40(6), 722 (2008)CrossRefPubMedPubMedCentralGoogle Scholar
- 3.Raphael, B., Volik, S., Yu, P., Wu, C., Huang, G., Waldman, F., Costello, J., Pienta, K., Mills, G., Bajsarowicz, K., Kobayashi, Y., Sridharan, S., Paris, P., Tao, Q., Aerni, S., Brown, R., Bashir, A., Gray, J., Cheng, J.F., Jong, P., Nefedov, M., Padilla-Nash, H., Collins, C.: A sequence based survey of the complex structural organization of tumor genomes. Genome Biology 9(3) (2008)Google Scholar
- 4.Ruan, Y., Ooi, H., Choo, S., Chiu, K., Zhao, X., Srinivasan, K., Yao, F., Choo, C., Liu, J., Ariyaratne, P., et al.: Fusion transcripts and transcribed retrotransposed loci discovered through comprehensive transcriptome analysis using Paired-End diTags (PETs). Genome Res. 17(6), 828–838 (2007)CrossRefPubMedPubMedCentralGoogle Scholar
- 5.Bashir, A., Volik, S., Collins, C., Bafna, V., Raphael, B.: Evaluation of Paired-End Sequencing Strategies for Detection of Genome Rearrangements in Cancer. PLoS Computational Biology 4(4) (2008)Google Scholar
- 6.Beigel, R., Alon, N., Apaydin, S., Fortnow, L., Kasif, S.: An Optimal Multiplex PCR Protocol for Closing Gaps in Whole Genomes. In: Proceedings of the Fifth Annual International Conference on Computational Molecular Biology (RECOMB) (2001)Google Scholar
- 7.Lipson, D.: Optimization problems in design of oligonucleotides for hybridization based methods. Master’s thesis, Technion - Israel Institute of Technology (2002)Google Scholar
- 10.Dasgupta, B., Jun, J., Mandoiu, I.: Primer Selection Methods for Detection of Genomic Inversions and Deletions via PAMP. In: Proceedings of the 6th Asia-Pacific Bioinformatics Conference. Imperial College Press (2008)Google Scholar
- 19.Kitagawa, Y., Inoue, K., Sasaki, S., Hayashi, Y., Matsuo, Y., Lieber, M.R., Mizoguchi, H., Yokota, J., Kohno, T.: Prevalent Involvement of Illegitimate V (D) J Recombination in Chromosome 9p21 Deletions in Lymphoid Leukemia. Journal of Biological Chemistry 277(48), 46289–46297 (2002)CrossRefPubMedGoogle Scholar
- 21.Lu, Q., Nunez, E., Lin, C., Christensen, K., Downs, T., Carson, D., Wang-Rodriguez, J., Liu, Y.: A sensitive array-based assay for identifying multiple TMPRSS2: ERG fusion gene variants. Nucleic Acids Research (2008)Google Scholar