High-throughput avian molecular sexing by SYBR green-based real-time PCR combined with melting curve analysis

• Main Authors: Chou Yii-Cheng, Wen Cheng-Hao, Su San-Hua, Chang Chia-Che, Gu De-Leung, Cheng Chun-An, Chang Hsueh-Wei, Chou Ta-Ching, Yao Cheng-Te, Tsai Chi-Li, Cheng Chien-Chung
• Format: Article
• Language: English
• Published: BMC 2008-02-01
• Series: BMC Biotechnology
• Online Access: http://www.biomedcentral.com/1472-6750/8/12

<p>Abstract</p> <p>Background</p> <p>Combination of <it>CHD </it>(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 <it>CHD-Z </it>and <it>CHD-W </it>genes is too short to be resolved.</p> <p>Results</p> <p>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. <it>Spilornis cheela hoya </it>(<it>S. c. hoya</it>) and <it>Pycnonotus sinensis </it>(<it>P. sinensis</it>) were used to illustrate this novel molecular sexing technique. The difference in the length of <it>CHD </it>genes in <it>S. c. hoya </it>and <it>P. sinensis </it>is 13-, and 52-bp, respectively. Using Griffiths' P2/P8 primers, molecular sexing failed both in PCR/electrophoresis of <it>S. c. hoya </it>and in PCR/MCA of <it>S. c. hoya </it>and <it>P. sinensis</it>. In contrast, we redesigned sex-specific primers to yield 185- and 112-bp PCR products for the <it>CHD-Z </it>and <it>CHD-W </it>genes of <it>S. c. hoya</it>, respectively, using PCR/MCA. Using this specific primer set, at least 13 samples of <it>S. c. hoya </it>were examined simultaneously and the Tm peaks of <it>CHD-Z </it>and <it>CHD-W </it>PCR products were distinguished.</p> <p>Conclusion</p> <p>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.</p>