一种基于聚合酶链反应（PCR）的快速简便方法，用于利用有限量的鳍组织分析转基因鱼。

A rapid and simple PCR-based method for analysis of transgenic fish using a restricted amount of fin tissue.

作者信息

Nam Yoon Kwon, Park Ji Eun, Kim Kyoung Kil, Kim Dong Soo

机构信息

Fish Genetic Manipulation Laboratory CNR), Department of Aquaculture, Pukyong National University, Pusan 608-737, South Korea.

出版信息

Transgenic Res. 2003 Aug;12(4):523-5. doi: 10.1023/a:1024274508052.

DOI:10.1023/a:1024274508052

PMID:12885174

Abstract

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.

摘要

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。