title: High-throughput sex identification by melting curve analysis in blue-breasted quail and chicken.
abstract: The objective was to develop a high-throughput method of identifying sex in both Coturnix chinensis and Gallus gallus, which would be useful for biomedical research and hatcheries. Because chromo-helicase-DNA binding protein (CHD)-based Griffiths P2/P8 primers do not produce polymerase chain reaction (PCR) products with distinguishable sex-specific curves in melting curve analysis (MCA), these primers are unsuitable for high throughput application in either species. Conserved regions were identified by basic local alignment search tool (BLAST) analyses of cloned CHD-Z and CHD-W genes of C. chinensis. Based on sequence alignment, a female-specific CHD-W primer (W-cot-F1) and a female/male (or CHD-W/CHD-Z)-common primer (ZW-cot-F1) were redesigned for use in combination with the Griffiths P2 primer for MCA-based PCR reaction. In C. chinensis and G. gallus, W-cot-F1/P2 and ZW-cot-F1/P2 had amplicon lengths of 315/318 and 114 base pairs and melting temperatures (Tm) of approximately 79.5 °C to 80 °C and approximately 78.5 °C to 79°C, respectively. Thus, MCA distinguished sex based on two distinct Tm peaks in females versus only one Tm peak in males. The MCA-based real-time PCR combined with the proposed primer redesign provided a high-throughput method of identifying sex in C. chinensis and G. gallus.
authors.affiliation: Department of Biomedical Science and Environmental Biology, Kaohsiung Medical University, Kaohsiung, Taiwan.
authors.forename: C-C
authors.initials: CC
authors.lastname: Chen
authors.forename: Y-S
authors.initials: YS
authors.lastname: Liu
authors.forename: C-C
authors.initials: CC
authors.lastname: Cheng
authors.forename: C-L
authors.initials: CL
authors.lastname: Wang
authors.forename: M-H
authors.initials: MH
authors.lastname: Liao
authors.forename: C-N
authors.initials: CN
authors.lastname: Tseng
authors.forename: H-W
authors.initials: HW
authors.lastname: Chang
mesh_terms: D000818:Animals; D030161:Avian Proteins / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D002645:Chickens / Q000502:physiology*; D003001:Cloning, Molecular; D017931:DNA Primers; D004268:DNA-Binding Proteins; D005260:Female; D005786:Gene Expression Regulation; D008297:Male; D009691:Nucleic Acid Denaturation; D011784:Quail / Q000502:physiology*; D017422:Sequence Analysis, DNA; D012732:Sex Determination Analysis / Q000379:methods / Q000662:veterinary*
chemical_list: D030161:Avian Proteins; D017931:DNA Primers; D004268:DNA-Binding Proteins