地高辛随机引物法标记探针的Southern杂交技术优化

›› 2009, Vol. 11 ›› Issue (4): 123-128.

地高辛随机引物法标记探针的Southern杂交技术优化

周长发^1,2,张锐¹,张晓¹,罗淑萍²,郭三堆¹

1.中国农业科学院生物技术研究所, 农作物基因资源与基因改良国家重大科学工程, 北京 100081; 2.新疆农业大学农学院, 乌鲁木齐 830052

收稿日期:2009-04-29 修回日期:2009-06-30 出版日期:2009-08-15 发布日期:2009-07-24
通讯作者: 郭三堆，研究员，主要从事棉花基因工程研究。E-mail：gsdui@mail.caas.net.cn
作者简介:周长发,硕士研究生,主要研究方向为植物基因工程。E-mail：zhou.chang.fa@163.com
基金资助:
国家转基因生物新品种培育科技重大专项（2008ZX08005-004）资助。

Technological Improvement of Southern Blot Using Digoxigenin-labeled Probes with Random-primed Method

ZHOU Zhang-fa ^1,2, ZHANG Rui ¹, ZHANG Xiao ², LUO Shu-ping ², GUO San-dui ¹

1.Biotechnology Research Institute, National Key Facility of Crop Gene Resources and Genetic Improvement,
Chinese Academy of Agricultural Sciences, Beijing 100081|2.Xinjiang Agricultural University, College of Agriculture, Urumqi 830052,China

Received:2009-04-29 Revised:2009-06-30 Online:2009-08-15 Published:2009-07-24

摘要/Abstract

摘要：

为检测转基因抗虫棉中的Bt Cry1A基因的表达，采用地高辛随机引物法标记探针、化学发光检测的Southern杂交技术，通过纯化探针、使用最适探针浓度、优化真空转印、杂交及免疫检测方法等一系列优化过程，最终得到背景低、信号强的Southern杂交结果。

关键词: 地高辛;化学发光法检测;转基因作物

Abstract:

Southern blot using digoxigenin(DIG)-labeled probes with random-primed method and chemiluminescent detection was used to detect Bt Cry1A gene of transgenic insect-resistant cotton. A serial of procedure was modified by purifying the DIG-labeled probes, using optimum probes concentration, improving the process of vacuum transferring, hybridization and immunoassay. Finally, we achieved good Southern blot result with low background and high sensitivity.

Key words: digoxigenin, chemiluminescent detection, transgenic plant

中图分类号:

Q781

周长发,张锐,张晓,罗淑萍,郭三堆. 地高辛随机引物法标记探针的Southern杂交技术优化[J]. , 2009, 11(4): 123-128.

ZHOU Zhang-fa, ZHANG Rui, ZHANG Xiao, LUO Shu-ping, GUO San-dui. Technological Improvement of Southern Blot Using Digoxigenin-labeled Probes with Random-primed Method[J]. , 2009, 11(4): 123-128.

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