期刊
ANALYTICAL BIOCHEMISTRY
卷 400, 期 1, 页码 69-77出版社
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.ab.2009.12.023
关键词
Preimplantation genetic diagnosis; Fluorescence resonance energy transfer; Hybridization probes; Melting curve analysis; Real-time PCR assay; beta-Thalassemia; HBB gene; Primer extension minisequencing
资金
- National Science Council of Taiwan [NSC 95-2314-B-002-280-MY3, NSC 96-2628-B-002-063-MY3]
Preimplantation genetic diagnosis (PGD) is employed increasingly to allow transfer of embryos to the uterus in assisted reproduction procedures. There are three stages of biopsy: polar bodies, one or two blastomeres from the cleavage-stage embryos, and trophectoderm cells (similar to 5 cells) from the blastocyst-stage embryos. Validation of polymerase chain reaction (PCR)-based assays are challenging because only limited genetic material can be obtained for PGD. In the current study, we modified a valid single-cell PCR protocol for PGD using real-time PCR assay with fluorescence resonance energy transfer (FRET) hybridization probes followed by melting curve analysis. We optimized and clinically applied the protocol, permitting molecular genetic analysis to amplify a specific region on the beta-globin (HBB) gene for a couple, carriers of two mutations: c.-78A>G and c.52A>T. Among a total of eight embryos obtained after ovarian stimulation, a single blastomere per embryo at the six- to eight-cell stage was biopsied. This PGD method showed that four embryos were unaffected, two embryos were selected for transfer, and one pregnancy was achieved. Finally, a healthy male baby was delivered at 38 weeks' gestation. The results obtained using the new method, FRET hybridization probes, were compared with findings using an existing method, primer extension minisequencing. (C) 2009 Elsevier Inc. All rights reserved.
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