Preimplantation genetic diagnosis of beta-thalassemia using real-time polymerase chain reaction with fluorescence resonance energy transfer hybridization probes.
Anal Biochem
; 400(1): 69-77, 2010 May 01.
Article
em En
| MEDLINE
| ID: mdl-20035706
ABSTRACT
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 ( approximately 5cells) 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 38weeks' gestation. The results obtained using the new method, FRET hybridization probes, were compared with findings using an existing method, primer extension minisequencing.
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Sondas de DNA
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Reação em Cadeia da Polimerase
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Talassemia beta
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Transferência Ressonante de Energia de Fluorescência
Tipo de estudo:
Diagnostic_studies
/
Guideline
Limite:
Adult
/
Female
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Humans
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Male
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Pregnancy
Idioma:
En
Ano de publicação:
2010
Tipo de documento:
Article