Snabes M C, Chong S S, Subramanian S B, Kristjansson K, DiSepio D, Hughes M R
Institute for Molecular Genetics, Baylor College of Medicine, Houston, TX 77030.
Proc Natl Acad Sci U S A. 1994 Jun 21;91(13):6181-5. doi: 10.1073/pnas.91.13.6181.
Due to the limited amount of DNA in a single diploid cell, preimplantation genetic diagnosis has relied on single- or dual-locus analyses in biopsied blastomers. We have applied single-cell whole-genome preamplification to PCR-based analysis of multiple disease loci from the same diploid cell. This method allows diagnosis of multiple disease genes, analysis of multiple exons/introns within a gene, or corroborative embryo-sex assignment and specific mutation detection at sex-linked loci. A blinded study of six genetic loci was performed with whole-genome preamplification followed by nested PCR. Amplification was observed in 103 of 105 assays (98%) and a correct diagnosis was made in 98%. All human blastomeres were correctly diagnosed (100%) at loci where the genotype could be confirmed, attesting to the reliability of the technique. Preamplification has now been applied successfully to the analysis of the two major mutations responsible for Tay-Sachs disease and of a common restriction polymorphism in the gene responsible for hemophilia A. The fidelity and length of product derived from this preamplification step make it an appealing technique for preimplantation genetic diagnoses requiring analyses at more than one locus.
由于单个二倍体细胞中的DNA数量有限，植入前基因诊断依赖于对活检的卵裂球进行单基因座或双基因座分析。我们已将单细胞全基因组预扩增应用于对来自同一二倍体细胞的多个疾病基因座进行基于PCR的分析。该方法可诊断多个疾病基因，分析基因内的多个外显子/内含子，或进行确证性胚胎性别鉴定以及检测性连锁基因座处的特定突变。对六个基因座进行了一项盲法研究，采用全基因组预扩增，随后进行巢式PCR。在105次检测中有103次（98%）观察到扩增，正确诊断率为98%。在可以确认基因型的基因座上，所有人类卵裂球均被正确诊断（100%），证明了该技术的可靠性。预扩增现已成功应用于分析导致泰-萨克斯病的两个主要突变以及导致甲型血友病的基因中的一个常见限制性多态性。该预扩增步骤产生的产物的保真度和长度使其成为一种有吸引力的技术，适用于需要在多个基因座进行分析的植入前基因诊断。
