Conditions of Chelex 100 Extraction for Feather DNA and Cloning of Sex-Specific DNA Sequences in Myiopsitta monachus for Sexing

• Main Author: 吳俊成
• Other Authors: Yan-Ming Horng
• Format: Others
• Language: zh-TW
• Online Access: http://ndltd.ncl.edu.tw/handle/70193912512547549059

碩士 === 國立嘉義大學 === 動物科學系研究所 === 96 === 英 文 摘 要 Abstract This study was conducted in two parts. The first part was intended to investigate the condition of the use of Chelex 100 for extracting DNA from an avian feather. The second part focused on investigating the sex sequences of Myiopsitta monachus, using random primers for random amplified polymorphic DNA (RAPD) fingerprinting analysis. In part one, five treatments were experimentally applied. They were 1 to 5 feather calamus; 5, 10, 15, 20% Chelex 100; heating to 56℃ for 1 to 11 hours; boiling for 1 to 10 minutes and the storage of the feathers for 1 to 20 weeks at room temperature. The results indicated that all treatments both efficiently extracted genomic DNA and yielded the signal from PCR amplification. To reduce the experimental time and cost, the conditions of 1 feather calamus, 5 % Chelex 100, heating at 56℃ for 1 hour and boiling for 8 minutes were utilized to extract feather DNA. In part two, a random primer, OPX-17, could amplify a fragment of approximately 800 bp for all females of Myiopsitta monachus. The sex-specific fragment was inserted into the vector and the sequence was determined. According to the sex-specific fragment, a pair of sex-specific primers, MonkSexX17 -F and MonkSexX17-R, were designed for PCR analysis. The results indicated that could amplify a 1143 bp fragment from all males/females of Myiopsitta monachus, but could only amplify a 800 bp specific fragment for females. The pair sex-specific primers can thus be used to identify the sex of Myiopsitta monachus.