Nam Yoon Kwon, Park Ji Eun, Kim Kyoung Kil, Kim Dong Soo
Fish Genetic Manipulation Laboratory CNR), Department of Aquaculture, Pukyong National University, Pusan 608-737, South Korea.
Transgenic Res. 2003 Aug;12(4):523-5. doi: 10.1023/a:1024274508052.
The protocol described in this paper offers a simple and rapid method for PCR analysis of transgenes using a restricted amount of fin tissue from small-sized transgenic fish. A simple preparation of fin lysate using a buffer containing a low concentration of an ionic detergent, SDS (0.01%), followed by neutralization with a second buffer containing higher concentrations of non-ionic detergents NP40 (2%) and Tween 20 (2%) consistently provides a reliable quantity of high-quality DNA template for PCR amplification of transgenes. Based on this protocol, transgenic fish can be clearly distinguished from non-transgenic fish using PCR in a rapid and reproducible manner. Tedious DNA purifications are avoided while fidelity of amplification and efficient identification of transgenic fish are maintained.
本文所述方案提供了一种简单快速的方法，可利用少量小型转基因鱼的鳍组织进行转基因的PCR分析。使用含有低浓度离子去污剂SDS（0.01%）的缓冲液简单制备鳍裂解物，随后用含有较高浓度非离子去污剂NP40（2%）和吐温20（2%）的第二种缓冲液进行中和，始终能为转基因的PCR扩增提供可靠数量的高质量DNA模板。基于此方案，可通过PCR以快速且可重复的方式清晰区分转基因鱼和非转基因鱼。避免了繁琐的DNA纯化过程，同时保持了扩增的保真度和转基因鱼的高效鉴定。
