Suazo A, Hall H G
University of Florida, Gainesville 32611, USA.
Biotechniques. 1999 Apr;26(4):704-5, 708-9. doi: 10.2144/99264st07.
The established amplified fragment-length polymorphism (AFLP) protocol was simplified and optimized for honey bee DNA (Apis mellifera L.). Compared to the original method, the following simplifications were made: (i) the digestion of DNA and ligation of the adapters are performed in one reaction vs. two, (ii) one restriction enzyme is used vs. two and (iii) amplification is accomplished in one reaction vs. two. PCR products are resolved in agarose-Synergel instead of polyacrylamide and are visualized by ethidium bromide staining rather than by autoradiography of labeled primers. Using the modified procedure for honey bee DNA, high reproducibility of the band patterns of PCR products and low sensitivity to the amplification conditions were seen. Analysis of honey bee DNA revealed considerable genetic variability within and between African and European bee samples. African- and European-specific fragments were found.
已建立的扩增片段长度多态性（AFLP）方案针对蜜蜂DNA（意大利蜜蜂）进行了简化和优化。与原始方法相比，进行了以下简化：（i）DNA消化和接头连接在一个反应中进行，而不是两个反应；（ii）使用一种限制性内切酶，而不是两种；（iii）扩增在一个反应中完成，而不是两个反应。PCR产物在琼脂糖-协同凝胶中分离，而不是在聚丙烯酰胺中，并且通过溴化乙锭染色进行可视化，而不是通过标记引物的放射自显影。使用针对蜜蜂DNA的改进程序，观察到PCR产物条带模式具有高重现性，并且对扩增条件的敏感性较低。对蜜蜂DNA的分析揭示了非洲蜜蜂和欧洲蜜蜂样本内部以及之间存在相当大的遗传变异性。发现了非洲和欧洲特有的片段。
