Journal
THERIOGENOLOGY
Volume 75, Issue 8, Pages 1543-1549Publisher
ELSEVIER SCIENCE INC
DOI: 10.1016/j.theriogenology.2010.12.018
Keywords
DNA amplification; GenomiPhi; Multiple displacement amplification (MDA); Phi29 DNA polymerase; Porcine embryo
Categories
Funding
- Japan Society for the Promotion of Science
- Research on Health Sciences focusing on Drug Innovation (Japan)
- The Ministry of Education, Science, Sports, and Culture, Japan
- Japan Science and Technology Agency
- Grants-in-Aid for Scientific Research [22580321, 23700562] Funding Source: KAKEN
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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. (C) 2011 Elsevier Inc. All rights reserved.
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