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P20: Improving Whole Genome Amplification methods for pre-implantation diagnosis
P.R. Nilsson et al. / European Journal of Medical Genetics 48 (2005) 443–476
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cation protocols. Other important features of the presented strategy are the inherent capacity to screen at significantly higher resolution enabling the screening of single exon deletions, the possibility to use the technique for both gene copy number quantification as well as gene expression profiling, and the compatibility of the method with both glass microarray and polystyrene microbead platforms. To conclude, we present a new platform for gene copy number analysis of specific regions and gene expression profiling of subsets of genes with low cost, high resolution and sensitivity. These dedicated tests will prove valuable for the analysis of specific chromosomal regions in prenatal testing, genomic classification of tumours as well as gene expression profiling of diagnostic or prognostic marker genes.
P20: Improving Whole Genome Amplification methods for pre-implantation diagnosis P.R. Nilsson a, J. Knijnenburg b, D.R. Gutknecht c, M.J. Eleveld a, M. Poot a, H. Tanke b, K. Szuhai b, J.K. Ploos van Amstel a a Department of Medical Genetics, Utrecht University Medical Centre, Lundlaan 6, 3584 EA Utrecht, The Netherlands b Department of Molecular Cell Biology, Leiden University Medical Center; Wassenaarseweg 72, 2333 AL Leiden, The Netherlands c Department for Reproductive Medicine, Utrecht University Medical Centre, Lundlaan 6, 3584 EA Utrecht, The Netherlands Sixty percent of recognized spontaneous abortions appear to contain one or more chromosomal aberrations, which are believed to account for a great deal of embryo wastage. In in vitro fertilization (IVF) this is partially reflected in decreased implantation efficiency of the embryo. Screening a single blastomere by using interphase FISH to detect common aneuploidies (X, Y, 13, 18, 21) followed by elimination of aberrant embryo’s has lead to an increase in ongoing pregnancy success rate. As interphase FISH based detection of all chromosomes is technically demanding this approach is not robust enough for routine application. A genome-wide aneuploidy screening by using array-CGH could be used for this purpose. Since a single cell does contain a limited amount of DNA, a whole genome amplification (WGA) step is needed to generate enough DNA to perform array-CGH. WGA has been carried out by DOP-PCR, but this method preferentially amplified certain regions of the human genome at the expense of others. Recently, two new strategies for WGA aiming at overcoming this drawback of DOPPCR have been developed. To determine whether these novel methods would be suitable for single cell WGA, we tested multiple displacement amplification (MDA; Qiagen) and the Omniplex WGA technology (Sigma). MDA is an isothermal amplification using bacteriophage phi29 DNA polymerase. Omniplex WGA technology uses in vitro libraries generated with fragmented DNA to amplify the entire genome by PCR. Since the MDA and the Omniplex kits are limited to 1 ng input DNA (1 cell contains ~ 7 pg), we tested several cell lysis procedures to improve DNA yield that is necessary for unbiased amplification. The amplified DNA was evaluated using STR- genotyping and array-CGH. Differences in yield and representation between the two WGA techniques were observed.
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cation protocols. Other important features of the presented strategy are the inherent capacity to screen at significantly higher resolution enabling the screening of single exon deletions, the possibility to use the technique for both gene copy number quantification as well as gene expression profiling, and the compatibility of the method with both glass microarray and polystyrene microbead platforms. To conclude, we present a new platform for gene copy number analysis of specific regions and gene expression profiling of subsets of genes with low cost, high resolution and sensitivity. These dedicated tests will prove valuable for the analysis of specific chromosomal regions in prenatal testing, genomic classification of tumours as well as gene expression profiling of diagnostic or prognostic marker genes.
P20: Improving Whole Genome Amplification methods for pre-implantation diagnosis P.R. Nilsson a, J. Knijnenburg b, D.R. Gutknecht c, M.J. Eleveld a, M. Poot a, H. Tanke b, K. Szuhai b, J.K. Ploos van Amstel a a Department of Medical Genetics, Utrecht University Medical Centre, Lundlaan 6, 3584 EA Utrecht, The Netherlands b Department of Molecular Cell Biology, Leiden University Medical Center; Wassenaarseweg 72, 2333 AL Leiden, The Netherlands c Department for Reproductive Medicine, Utrecht University Medical Centre, Lundlaan 6, 3584 EA Utrecht, The Netherlands Sixty percent of recognized spontaneous abortions appear to contain one or more chromosomal aberrations, which are believed to account for a great deal of embryo wastage. In in vitro fertilization (IVF) this is partially reflected in decreased implantation efficiency of the embryo. Screening a single blastomere by using interphase FISH to detect common aneuploidies (X, Y, 13, 18, 21) followed by elimination of aberrant embryo’s has lead to an increase in ongoing pregnancy success rate. As interphase FISH based detection of all chromosomes is technically demanding this approach is not robust enough for routine application. A genome-wide aneuploidy screening by using array-CGH could be used for this purpose. Since a single cell does contain a limited amount of DNA, a whole genome amplification (WGA) step is needed to generate enough DNA to perform array-CGH. WGA has been carried out by DOP-PCR, but this method preferentially amplified certain regions of the human genome at the expense of others. Recently, two new strategies for WGA aiming at overcoming this drawback of DOPPCR have been developed. To determine whether these novel methods would be suitable for single cell WGA, we tested multiple displacement amplification (MDA; Qiagen) and the Omniplex WGA technology (Sigma). MDA is an isothermal amplification using bacteriophage phi29 DNA polymerase. Omniplex WGA technology uses in vitro libraries generated with fragmented DNA to amplify the entire genome by PCR. Since the MDA and the Omniplex kits are limited to 1 ng input DNA (1 cell contains ~ 7 pg), we tested several cell lysis procedures to improve DNA yield that is necessary for unbiased amplification. The amplified DNA was evaluated using STR- genotyping and array-CGH. Differences in yield and representation between the two WGA techniques were observed.