Simplified DNA extraction methods for sexing chick embryos
Simplified DNA extraction methods for sexing chick embryos
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DOI：
10.2141/jpsa.41.147
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发表时间：
2004-04-01
影响因子：
1.5
通讯作者：
Kanai, Yukio
中科院分区：
文献类型：
--
作者：
Minematsu, Takeo;Sugiyama, Misato;Kanai, Yukio
我们研究了从成年鸡和早期鸡胚胎中提取DNA的效率和基于聚合酶链式反应(PCR)的性别鉴定的准确性。DNA样品的制备采用水、氨水和蛋白酶三种方法。通过扩增717个碱基的W染色体特异XhoI重复序列的276个碱基片段，进行基于PCR的性别鉴定。在成年鸡的血液样本中，上述三种方法提取DNA所需的时间比传统的苯酚/氯仿/异戊醇(PCI)方法所需的时间要短。使用氨法和蛋白酶法制备的DNA样本进行准确的基于PCR的性别鉴定是可能的。基于PCR的性别鉴定成功地从一个2天大的胚胎和一个7天大的胚胎的小肠、性腺和肾脏中提取了DNA样本，这些DNA样本是用氨法和蛋白酶法提取的。然而，从7日龄胚胎的性腺中提取的DNA不能用氨法进行可靠的性别鉴定。综上所述，氨法和蛋白酶法是一种简单实用的DNA提取方法，用于基于PCR的性别鉴定，但如果DNA样本以前没有用于性别鉴定，则应检查使用氨法后性别鉴定的可靠性。
We investigated the efficiency of DNA extraction and the accuracy of polymerase chain reaction- (PCR-) based sexing of DNA that was obtained from adult chickens and early chicken embryos. DNA samples were prepared using water-, ammonium-, and proteinase-mediated extraction. PCR-based sexing was performed by amplifying a 276 -bp fragment of the 717-bp W-chromosome-specific XhoI repetitive sequence. In blood samples from adult chickens, the time required for DNA extraction using the three aforementioned methods was less than that required by the conventional method of using phenol/chloroform/isoamyl alcohol (PCI). Accurate PCR-based sexing was possible using DNA samples that were prepared using the ammonium and proteinase methods. PCR-based sexing was successful for DNA samples from a 2-day-old embryo and the small intestine, gonad, and kidney of a 7-day-old embryo that were extracted using the ammonium and proteinase methods. However, DNA from the gonads of the 7-day-old embryo could not be sexed reliably using the ammonium method. In summary, the ammonium and proteinase methods are simple and practical means of extracting DNA for PCR-based sexing, but the reliability of sexing after use of the ammonium method should be checked when DNA samples have not been used previously for sexing.