Danek Gertner Institute of Human Genetics, Sheba Medical Center, Tel-Hashomer, Ramat Gan, 52621, Israel.
J Assist Reprod Genet. 2010 Jun;27(6):335-41. doi: 10.1007/s10815-010-9396-5. Epub 2010 Mar 3.
To investigate the influence of amplicons size and cell type on allele dropout and amplification failures in single-cell based molecular diagnosis.
730 single lymphocytes and amniotic cells were collected from known heterozygotes individuals to one of the common Ashkenazi Jewish mutations: 1278+TATC and IVS12+1G>C which cause Tay Sachs Disease, IVS20+6T and 854A>C which underlie Familial Dysautonomia and Canavan Disease. DNA was extracted and analyzed by our routine methods.
Reduced rates of allele dropout and amplification failure were found when smaller amplification product were designed and in amniotic cultured cells compared to peripheral lymphocytes. Cultured lymphocytes, induced to divide, demonstrated significantly less allele dropout than non induced lymphocytes suggesting the role of division potential on amplification efficiency.
Single cell based diagnosis should be designed for each mutation. Minimal sized amplicons and cell having division potential should be preferred, as well as sensitive techniques to detect preferential amplification.
研究扩增子大小和细胞类型对基于单细胞的分子诊断中等位基因缺失和扩增失败的影响。
从已知杂合子个体中收集了 730 个单个淋巴细胞和羊水细胞，这些个体携带有常见的阿什肯纳兹犹太突变之一：导致泰萨克斯病的 1278+TATC 和 IVS12+1G>C、导致家族性自主神经机能异常和 Canavan 病的 IVS20+6T 和 854A>C。提取 DNA 并通过我们的常规方法进行分析。
与外周淋巴细胞相比，设计较小的扩增产物和在羊水培养细胞中时，等位基因缺失和扩增失败的比率降低。与未诱导的淋巴细胞相比，诱导分裂的培养淋巴细胞的等位基因缺失明显减少，这表明分裂潜能对扩增效率的作用。
应根据每个突变设计基于单细胞的诊断。应优先选择最小大小的扩增子和具有分裂潜能的细胞，以及用于检测优先扩增的敏感技术。
