PREIMPLANTATION SINGLE-CELL ANALYSIS OF MULTIPLE GENETIC-LOCI BY WHOLE-GENOME AMPLIFICATION

Due to the Limited amount of DNA in a single diploid cell, preimplanta tion genetic diagnosis has relied on single- or dual-locus analyses in biopsied blastomeres. We have applied single-cell whole-genome preamp lification to PCR-based analysis of multiple disease loci from the sam e diploid cell. This method allows diagnosis of multiple disease genes , analysis of multiple exons/introns within a gene, or corrobo- rative embryo-sex assignment and specific mutation detection at sex-linked l oci. A blinded study of six genetic loci was performed with whole-geno me preamplification followed by nested PCR. Amplification was observed in 103 of 105 assays (98%) and a correct diagnosis was made In 98%. A ll human blastomeres were correctly diagnosed (100%) at loci where the genotype could be confirmed, attesting to the reliability of the tech nique. Preamplification has now been applied successfully to the analy sis of the two major mutations responsible for Tay-Sachs disease and o f a common restriction polymorphism in the gene responsible for hemoph ilia A. The fidelity and length of product derived from this preamplif ication step make it an appealing technique for preimplantation geneti c diagnoses requiring analyses at more than one locus.