Methodology article

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

Hsueh-Wei Chang^1,2 , Chun-An Cheng³ , De-Leung Gu¹ , Chia-Che Chang⁴ , San-Hua Su¹ , Cheng-Hao Wen¹ , Yii-Cheng Chou⁵ , Ta-Ching Chou⁶ , Cheng-Te Yao^7,8 , Chi-Li Tsai^7,9 and Chien-Chung Cheng¹

¹Faculty of Biomedical Science and Environmental Biology, Kaohsiung Medical University, Kaohsiung, Taiwan

²Graduate Institute of Natural Products, College of Pharmacy, Kaohsiung Medical University, Kaohsiung, Taiwan

³College of Life Science, National Tsing Hua University, HsinChu, Taiwan

⁴Institute of Biomedical Sciences, National Chung Hsing University, Taichung, Taiwan

⁵Institute of Biotechnology, Chung Hwa College of Medical Technology, Tainan, Taiwan

⁶Institute of Ecology and Evolutionary Biology, National Taiwan University, Taipei, Taiwan

⁷Taiwan Endemic Species Research Institute, Nantou, Taiwan

⁸Institute of Biodiversity, National Cheng Kung University, Tainan, Taiwan

⁹Department of Life Sciences, National Chung Hsing University, Taichung, Taiwan

author email corresponding author email

BMC Biotechnology 2008, 8:12doi:10.1186/1472-6750-8-12

Published:	12 February 2008

Abstract

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.