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Cloning the swamp buffalo SRY gene for embryo sexing with multiplex-nested PCR.
- Source :
-
Theriogenology [Theriogenology] 2007 Dec; Vol. 68 (9), pp. 1211-8. Date of Electronic Publication: 2007 Oct 24. - Publication Year :
- 2007
-
Abstract
- The polymerase chain reaction (PCR) is an efficient method for sexing embryos. The objective of this study was to develop an accurate and reliable method for sexing swamp buffalo (Bubalus bubalis) embryos. The SRY gene from swamp buffalo genomic DNA was amplified by PCR, using primers based on the sequence of the Holstein SRY gene. This fragment was sequenced based on a BLAST search; the SRY gene was highly conserved. Using a Southern blot, there was a strong signal in genomic DNA only from male swamp buffalo. Two pairs of nested primers, targeted to amplify the swamp buffalo SRY conserved region, were designed for sex identification. Simultaneously, the G3PDH gene was co-amplified to serve as an internal control. A multiplex-nested PCR system was optimized by varying the following individually: concentrations of Mg(2+) and dNTPs, ratio of concentrations of primers and numbers of cycles. Biopsies of 27 IVF-derived embryos and 24 embryos fertilized with Y-chromosome-bearing sperm were examined. Using optimized procedures, clear signals following PCR amplification were obtained from all embryo samples; PCR amplification accuracy was further verified by comparing PCR and dot blots. We concluded that this PCR technique was highly reliable for sexing swamp buffalo embryos.
- Subjects :
- Animals
Base Sequence
Blotting, Southern veterinary
Buffaloes genetics
Cloning, Molecular
DNA Primers chemistry
Embryo, Mammalian physiology
Female
Male
Molecular Sequence Data
Phylogeny
Polymerase Chain Reaction methods
Reproducibility of Results
Sequence Analysis, DNA
Sex Determination Analysis methods
Buffaloes embryology
Genes, sry genetics
Polymerase Chain Reaction veterinary
Sex Determination Analysis veterinary
Subjects
Details
- Language :
- English
- ISSN :
- 0093-691X
- Volume :
- 68
- Issue :
- 9
- Database :
- MEDLINE
- Journal :
- Theriogenology
- Publication Type :
- Academic Journal
- Accession number :
- 17928043
- Full Text :
- https://doi.org/10.1016/j.theriogenology.2007.07.007