F Jin1, H Huang, Y Ye. 1. Department of Obstetrics, Affiliated Obstetrics and Gynecology Hospital, Faculty of Medicine, Zhejiang University, Hangzhou 310006, China.
Abstract
OBJECTIVE: To investigate the uniformity of whole genome amplification from a single cell by degenerate oligonucleotide primed polymer ase chain reaction (DOP-PCR). METHODS: Amplification of the whole genomic DNA from a single cell with XX, XY, XO, XXY, +13 or +21 was performed by DOP-PCR. Comparative genomic hybridization (CGH) of a single cell DOP-PCR product against the genomic DNA or a single cell DOP-PCR product from normal male as carried out respectively. RESULTS: The average profile of fluorescence intensive ratio in CGH with the genomic DNA as the reference was greatly fluctuated and the standard deviation in about 30% haploid was beyond the normal limits. False positive hyperrepresentation was existed in X chromosome while trisomy 13 and 21 were failed to be detected. However, the distributions of the mean and the standard deviation of the ratio in the CGH with DOP-PCR product as the reference were acceptable. The copy number changes of chromosome X, Y, 13 and 21 were demonstrated. CONCLUSION: Although the single cell DOP-PCR product is not completely uniform, it would be reliable to study the whole genome of a single cell by controlling the conditions of PCR and selecting the suitable control reference because the unequal amplification is unrandom.
OBJECTIVE: To investigate the uniformity of whole genome amplification from a single cell by degenerate oligonucleotide primed polymer ase chain reaction (DOP-PCR). METHODS: Amplification of the whole genomic DNA from a single cell with XX, XY, XO, XXY, +13 or +21 was performed by DOP-PCR. Comparative genomic hybridization (CGH) of a single cell DOP-PCR product against the genomic DNA or a single cell DOP-PCR product from normal male as carried out respectively. RESULTS: The average profile of fluorescence intensive ratio in CGH with the genomic DNA as the reference was greatly fluctuated and the standard deviation in about 30% haploid was beyond the normal limits. False positive hyperrepresentation was existed in X chromosome while trisomy 13 and 21 were failed to be detected. However, the distributions of the mean and the standard deviation of the ratio in the CGH with DOP-PCR product as the reference were acceptable. The copy number changes of chromosome X, Y, 13 and 21 were demonstrated. CONCLUSION: Although the single cell DOP-PCR product is not completely uniform, it would be reliable to study the whole genome of a single cell by controlling the conditions of PCR and selecting the suitable control reference because the unequal amplification is unrandom.