Whole-genome amplification-based GenomiPhi for multiple genomic analysis of individual early porcine embryos

摘要

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, similar to 81% of the 103.5 ng (84 ng) of amplified DNA was estimated to be porcine sequences (2.2 X 10(3)-fold enrichment). In addition, PCR confirmed the presence of transgenes, as well as endogenous alpha-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