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標題: High-throughput avian molecular sexing by SYBR green-based real-time PCR combined with melting curve analysis
作者: Chang, H.W.
Cheng, C.A.
Gu, D.L.
Chang, C.C.
Su, S.H.
Wen, C.H.
Chou, Y.C.
Chou, T.C.
Yao, C.T.
Tsai, C.L.
Cheng, C.C.
關鍵字: polymerase chain-reaction;dna;identification;birds;expression;product;marker
Project: Bmc Biotechnology
期刊/報告no:: Bmc Biotechnology, Volume 8.
Background: Combination of CHD (chromo-helicase-DNA binding protein)- specific polymerase chain reaction (PCR) with electrophoresis (PCR/electrophoresis) is the most common avian molecular sexing technique but it is lab-intensive and gel-required. Gender determination often fails when the difference in length between the PCR products of CHD-Z and CHD-W genes is too short to be resolved. Results: Here, we are the first to introduce a PCR-melting curve analysis (PCR/MCA) to identify the gender of birds by genomic DNA, which is gel-free, quick, and inexpensive. Spilornis cheela hoya ( S. c. hoya) and Pycnonotus sinensis ( P. sinensis) were used to illustrate this novel molecular sexing technique. The difference in the length of CHD genes in S. c. hoya and P. sinensis is 13-, and 52-bp, respectively. Using Griffiths' P2/P8 primers, molecular sexing failed both in PCR/electrophoresis of S. c. hoya and in PCR/MCA of S. c. hoya and P. sinensis. In contrast, we redesigned sex-specific primers to yield 185- and 112-bp PCR products for the CHD-Z and CHD-W genes of S. c. hoya, respectively, using PCR/MCA. Using this specific primer set, at least 13 samples of S. c. hoya were examined simultaneously and the Tm peaks of CHD-Z and CHD-W PCR products were distinguished. Conclusion: In this study, we introduced a high-throughput avian molecular sexing technique and successfully applied it to two species. This new method holds a great potential for use in high throughput sexing of other avian species, as well.
ISSN: 1472-6750
DOI: 10.1186/1472-6750-8-12
Appears in Collections:生物醫學研究所

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