Matioli S R, de Brito R A
Dept. de Biologia, Instituto de Biociências, Universidade de São Paulo, Brazil.
Biotechniques. 1995 Nov;19(5):752-6, 758.
A technique to obtain genetic markers is described. PCR is performed at two different annealing temperatures in a single reaction mixture. During the first cycles, a single microsatellite oligonucleotide is used as primer to amplify DNA between two microsatellite regions. The annealing temperature and consequent stringency is then lowered, and in further cycles a random-amplified polymorphic DNA (RAPD) primer exponentially amplifies parts of the previously amplified fragments, which contain sequences complementary to the RAPD primer. The first round of specific amplification is found to be crucial for obtaining consistent, repeatable results, as compared with the results obtained by RAPD alone. This technique was proven to produce genetically informative markers, as revealed by crosses performed with Drosophila mercatorum strains, and it can be used for genetic mapping or population genetics studies.
本文描述了一种获取遗传标记的技术。在单一反应混合物中于两个不同的退火温度下进行聚合酶链式反应（PCR）。在最初的循环中，使用单个微卫星寡核苷酸作为引物来扩增两个微卫星区域之间的DNA。然后降低退火温度及相应的严谨性，在后续循环中，随机扩增多态性DNA（RAPD）引物对先前扩增片段的部分进行指数扩增，这些片段包含与RAPD引物互补的序列。与单独使用RAPD获得的结果相比，发现第一轮特异性扩增对于获得一致、可重复的结果至关重要。如通过与墨卡托果蝇（Drosophila mercatorum）菌株进行杂交所揭示的，该技术被证明能产生具有遗传信息的标记，并且可用于遗传图谱绘制或群体遗传学研究。
