PREIMPLANTATION DIAGNOSIS BY WHOLE-GENOME AMPLIFICATION, PCR AMPLIFICATION, AND SOLID-PHASE MINISEQUENCING OF BLASTOMERE DNA

We have developed a new method for preimplantation diagnosis of inheri ted diseases. Our procedure for the identification of point mutations in single cells combines whole-genome amplification using 15-mer rando m primers (primer extension preamplification, PEP) with a single locus -specific PCR amplification, followed by detection of the mutation by solid-phase minisequencing. The procedure was evaluated by detecting t hree disease-causing mutations and seven polymorphic nucleotides locat ed on different human chromosomes from single ganulosa and blastomere cells, The correct genotype of the cell was identified at 96% of the n ucleotide positions analyzed, showing that a representative part of th e genome is amplified during PEP. We estimate that PEP yielded at leas t 1000 copies of the genome. The quantitative nature of the solid-phas e minisequencing method allowed us to notice that preferential amplifi cation of one allele occurs at heterozygous loci during PEP, which is a potential problem in preimplantation diagnosis.