High-throughput applicable genomic sex typing of chicken by TaqMan real-time quantitative polymerase chain reaction

Genetic sex typing of vertebrate animals is an essential technique for research on reproductive phenomena such as sex determination of embryonic tissues. Polymerase chain reaction amplification of genomic DNA segments in the Z and W sex chromosomes has been widely used as a standard laboratory method to determine genetic sex of the chicken (Gallus gallus domesticus). However, conventional protocols for PCR determination of avian sex typically involve tedious steps of genomic DNA isolation, which often require relatively large amounts of tissue samples, and the purity of genomic DNA specimens significantly affects PCR efficiency. Moreover, detection of sex chromosome-specific PCR products by gel electrophoresis is prone to misjudgment caused by amplification of contaminating genomic DNA segments derived from tissue or DNA samples as well as previously generated PCR products. Thus, the credibility of genetic sex typing by conventional PCR-based methods that measure the relative amounts of the end product DNA amplicons critically depends on several experimental steps that are potentially vulnerable to errors. Here, we describe an optimized protocol of chicken genetic sex typing by TaqMan real-time quantitative PCR amplification of markers on the sex chromosomes. This TaqMan sex typing method accurately quantifies relative amounts of the Z and W sex chromosome markers directly from only 0.5 to 2 microL of total blood lysate without nucleic acid purification. The real-time amplification curves of the quantitative PCR reaction readily distinguished truly homozygous (ZZ) and heterozygous (ZW) sex chromosomes from contamination of the sex chromosomal DNA, ensuring highly credible sex determination. Thus, the TaqMan typing of chicken genetic sex has several advantageous features for high-throughput operation compared with conventional methods.