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Volume 63 Number 1 >

Genetic diagnosis of band 3 deficiency using a quenching probe (QProbe)-PCR assay in bovine embryos

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Title: Genetic diagnosis of band 3 deficiency using a quenching probe (QProbe)-PCR assay in bovine embryos
Authors: Kageyama, Soichi Browse this author
Hirayama, Hiroki Browse this author →KAKEN DB
Moriyasu, Satoru Browse this author
Minamihashi, Akira Browse this author
Keywords: Band 3
Preimplantation genetic diagnosis
quenching probe
Issue Date: Feb-2015
Publisher: Graduate School of Veterinary Medicine, Hokkaido University
Journal Title: Japanese Journal of Veterinary Research
Volume: 63
Issue: 1
Start Page: 45
End Page: 51
Abstract: The present study was conducted to develop a simple and rapid procedure to determine the genotype of band 3 deficiency in bovine embryos by a novel real-time PCR method using a fluorescent quenching-based probe (QProbe-PCR). QProbe-PCR successfully distinguished wild type and R664X mutant alleles by melting curve analysis. Minimal amounts of DNA template were required for the detection of wild type/wild type alleles, mutant/mutant alleles, and wild type/mutant alleles; their amounts were 10 pg, 25 pg, and 50 pg, respectively. When 10 blastomeres were used as a DNA sample, accuracies of genotyping by QProbe-PCR were 100% and 89% in embryos homozygous for the wild type allele and heterozygous for the wild type and mutant alleles, respectively. QProbe-PCR takes approximately 2 h for genotyping and requires lesser time than the conventional method using PCR-RFLP, which requires digestion with a restriction enzyme and electrophoresis. Our data showed that QProbe-PCR is a useful method for rapid analysis of the genetic deficiency in preimplantation embryos. Reduction in the time required for genotyping enabled the transfer of genetically selected embryos to recipient cows on the day of embryo collection. These results suggest that determination of the genotype for the genetic deficiency in embryos is useful to select animals free from the genetic disease, and it also makes it possible to produce an animal model homozygous for the mutation.
Type: bulletin (article)
Appears in Collections:Japanese Journal of Veterinary Research > Volume 63 Number 1

Submitter: 獣医学部図書室

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