Multiple displacement amplification on single cell and possible PGD applications
Multiple displacement amplification on single cell and possible PGD applications
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DOI：
10.1093/molehr/gah114
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发表时间：
2004-11-01
影响因子：
4
通讯作者：
Ozand, P
中科院分区：
文献类型：
--
作者：
Hellani, A;Coskun, S;Ozand, P
多重置换扩增 (MDA) 是一种用于扩增极少量 DNA 的技术，据报道可以产生大量高质量 DNA。我们使用 MDA 直接从单个细胞扩增整个基因组。 PGD 最常用的技术是 PCR 和荧光原位杂交 (FISH)。这些技术有许多限制，包括 FISH 诊断的染色体数量或单细胞 PCR 产生的 DNA 的质量。该报告首次展示了使用 MDA 进行单细胞全基因组扩增。总共 16 个短串联重复序列 (STR) 被成功扩增，其模式与基因组 DNA 相似。此外，通过分析 (i) 已知 β 珠蛋白突变 (IVSI-5 C-G) 的杂合性和研究 (ii) STR 中存在的杂合位点，在 40 个单细胞中评估了源自 MDA 的等位基因缺失 (ADO)。 β 珠蛋白基因测序的 ADO 为 10.25%，STR 荧光 PCR 分析的 ADO 为 5%。此外，MDA 的扩增精度允许使用比较基因组杂交阵列检测单个细胞上的 21 三体性。总之，这些数据表明 MDA 可用于单细胞分子核型分析和 PGD 中任何单基因疾病的诊断。
Multiple displacement amplification (MDA) is a technique used in the amplification of very low amounts of DNA and reported to yield large quantities of high-quality DNA. We used MDA to amplify the whole genome directly from a single cell. The most common techniques used in PGD are PCR and fluorescent in-situ hybridization (FISH). There are many limitations to these techniques including, the number of chromosomes diagnosed for FISH or the quality of DNA issued from a single cell PCR. This report shows, for the first time, use of MDA for single cell whole genome amplification. A total of 16 short tandem repeats (STRs) were amplified successfully with a similar pattern to the genomic DNA. Furthermore, allelic drop out (ADO) derived from MDA was assessed in 40 single cells by analysing (i) heterozygosity for a known beta globin mutation (IVSI-5 C-G) and by studying (ii) the heterozygous loci present in the STRs. ADO turned out to be 10.25% for the beta globin gene sequencing and 5% for the fluorescent PCR analysis of STRs. Moreover, the amplification accuracy of MDA permitted the detection of trisomy 21 on a single cell using comparative genome hybridization-array. Altogether, these data suggest that MDA can be used for single cell molecular karyotyping and the diagnosis of any single gene disorder in PGD.