title: Preimplantation diagnosis by whole-genome amplification, PCR amplification, and solid-phase minisequencing of blastomere DNA.
abstract: We have developed a new method for preimplantation diagnosis of inherited diseases. Our procedure for the identification of point mutations in single cells combines whole-genome amplification using 15-mer random 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 three disease-causing mutations and seven polymorphic nucleotides located on different human chromosomes from single granuloma and blastomere cells. The correct genotype of the cell was identified at 96% of the nucleotide positions analyzed, showing that a representative part of the genome is amplified during PEP. We estimate that PEP yielded at least 1000 copies of the genome. The quantitative nature of the solid-phase minisequencing method allowed us to notice that preferential amplification of one allele occurs at heterozygous loci during PEP, which is a potential problem in preimplantation diagnosis.
authors.affiliation: Department of Human Molecular Genetics, National Public Health Institute, Helsinki, Finland.
authors.forename: T
authors.initials: T
authors.lastname: Paunio
authors.forename: I
authors.initials: I
authors.lastname: Reima
authors.forename: A C
authors.initials: AC
authors.lastname: Syvänen
mesh_terms: D000483:Alleles; D001483:Base Sequence; D001757:Blastomeres; D004247:DNA; D004252:DNA Mutational Analysis; D047108:Embryonic Development / Q000235:genetics; D005260:Female; D005784:Gene Amplification; D006580:Genetic Carrier Screening; D005838:Genotype; D006107:Granulosa Cells / Q000737:chemistry; D006801:Humans; D008969:Molecular Sequence Data; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D011110:Polymorphism, Genetic; D011247:Pregnancy; D011296:Prenatal Diagnosis / Q000379:methods*
chemical_list: D004247:DNA