Zoo Biology 27:62–69 (2008) BRIEF REPORT An Evaluation of CHD-Specific Primer Sets for Sex Typing of Birds from Feathers Alan H.K. Ong 1Ã and Subramaniam Vellayan 2 1 Department of Bioscience and Chemistry, Universiti Tunku Abdul Rahman, Kuala Lumpur, Malaysia 2 Zoo Negara Malaysia, Selangor, Malaysia The amplification of the highly conserved chromo-helicase-DNA binding region found in both the Z and W chromosome was evaluated with three sets of primers (P8/P2, 1237L/1272H and 2550F/2718R). DNA extracted from feathers through a simple boiling method was used to address its reliability in generating the sex- linked bands. All the bird samples, including the seven bird families that have not been reported previously, were successfully amplified with the primer set 2550F/ 2718R. The resulting polymerase chain reaction products showed clearly resolved fragments on a conventional agarose gel electrophoresis with size differences ranging from 80 to 540 bp between the two respective ZW gene copies. Although the P8/P2 primer was not as effective under the same conditions, it was able to produce well-resolved Z and W bands from bird species under the Antidea family, whereas the 2250F/2718R primer set only produced a single amplified fragment of a different size between the male and the female. Zoo Biol 27:62–69, 2008.  c 2007 Wiley-Liss, Inc. Keywords: molecular bird sexing; ZW intron regions; NaOH boiling method Published online 2 January 2008 in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/zoo.20163 Received 6 December 2006; Revised 4 October 2007; Accepted 30 October 2007 Grant sponsor: Universiti Tunku Abdul Rahman; Grant number: IPSR/UTARRF(A)/05/L03. Ã Correspondence to: Alan Han Kiat Ong, Department of Bioscience and Chemistry, Faculty of Engineering and Science, Universiti Tunku Abdul Rahman, Jln. Genting Kelang, Setapak, 53300, Kuala Lumpur, Malaysia. E-mail: onghk@mail.utar.edu.my r r 2007 Wiley-Liss, Inc.