title: Whole-genome amplification-based GenomiPhi for multiple genomic analysis of individual early porcine embryos.
abstract: The multiple displacement amplification (MDA) method, which relies on isothermal DNA amplification using the DNA polymerase of the bacteriophage phi29, was recently developed for high-performance, whole-genome amplification (WGA). The objective of the present study was to determine whether a target sequence could be successfully amplified by conventional PCR when the genomic DNA of a single Day-7 porcine blastocyst (derived from SCNT of a gene-engineered fibroblast) was amplified by the MDA method and used as a template. The yield of double-stranded DNA was 103.5 ± 16.0 ng/embryo (range, 75-125), as assessed by a PocoGreen assay. However, non-specific products (20 ± 5 ng/tube) were also generated, even in the negative control. Thus, ∼81% of the 103.5 ng (84 ng) of amplified DNA was estimated to be porcine sequences (2.2 × 10(3)-fold enrichment). In addition, PCR confirmed the presence of transgenes, as well as endogenous α-1,3-galactosyltransferase and homeobox Nanog genes in all embryos. Sequencing of the amplified products verified the fidelity of this system. In conclusion, the MDA-mediated WGA, which was simple, inexpensive, and did not require a thermal cycler, could be a powerful tool for multiple genomic analyses of individual early porcine embryos.
authors.affiliation: Kagoshima University, Kagoshima, Kagoshima 890-0065, Japan.
authors.forename: Eri
authors.initials: E
authors.lastname: Akasaka
authors.forename: Akio
authors.initials: A
authors.lastname: Ozawa
authors.forename: Hironori
authors.initials: H
authors.lastname: Mori
authors.forename: Yamato
authors.initials: Y
authors.lastname: Mizobe
authors.forename: Mitsutoshi
authors.initials: M
authors.lastname: Yoshida
authors.forename: Kazuchika
authors.initials: K
authors.lastname: Miyoshi
authors.forename: Masahiro
authors.initials: M
authors.lastname: Sato
mesh_terms: D000818:Animals; D001755:Blastocyst / Q000378:metabolism; D004247:DNA; D018507:Gene Expression Regulation, Developmental; D016678:Genome; D023281:Genomics / Q000379:methods*; D021141:Nucleic Acid Amplification Techniques / Q000379:methods / Q000662:veterinary*; D013552:Swine / Q000196:embryology* / Q000235:genetics*
chemical_list: D004247:DNA