Bonnema G, Hontelez J, Verkerk R, Zhang Y Q, van Daelen R, van Kammen A, Zabel P
Department of Molecular Biology, Agricultural University, Wageningen, The Netherlands.
Plant J. 1996 Jan;9(1):125-33. doi: 10.1046/j.1365-313x.1996.09010125.x.
An improved method for preparing partially digested tomato DNA has been developed, that is suitable for YAC cloning. It involves (i) isolation of high molecular-weight DNA from agarose-embedded leaf protoplasts, (ii) controlled partial digestion in situ using EcoRl endonuclease in the presence of EcoRI methylase (M. EcoRl), and (iii) fractionation of the partial digest on a Clamped Homogeneous Electric Fields (CHEF) gel. Unlike methods commonly used for generating partial digests, the present method allows one to produce digests in which the bulk of restriction fragments are of the desired size. Use of these partial digests in constructing YAC libraries of the tomato lines Moneymaker-Cf4 and VFNT Cherry resulted in libraries (total 21,060 clones, 5.5 genome equivalents) in which 80% of the YACs have inserts between 200 and 600 kb. Both libraries have been screened with selected RFLP markers linked to the Cladosporium fulvum Cf4 locus on chromosome 1, using a three-dimensional PCR-based screening technique. To this end, the RFLP markers have been sequenced to allow for the synthesis of specific primers. Thus, for each marker tested several YAC clones have been isolated, including a family of clones that carry leucine-rich repeat sequences located around the Cf4/Cf9 locus.
已开发出一种改进的制备部分消化番茄DNA的方法，该方法适用于酵母人工染色体（YAC）克隆。它包括：（i）从琼脂糖包埋的叶原生质体中分离高分子量DNA；（ii）在EcoRI甲基化酶（M.EcoRI）存在的情况下，使用EcoRI内切酶进行原位控制部分消化；（iii）在钳位均匀电场（CHEF）凝胶上对部分消化产物进行分级分离。与通常用于产生部分消化的方法不同，本方法能够产生大部分限制性片段为所需大小的消化产物。将这些部分消化产物用于构建番茄品系Money maker-Cf4和VFNT Cherry的YAC文库，得到了文库（共21,060个克隆，5.5个基因组当量），其中80%的YAC插入片段在200至600 kb之间。使用基于三维PCR的筛选技术，用与1号染色体上番茄叶霉病Cf4基因座连锁的选定RFLP标记对这两个文库进行了筛选。为此，对RFLP标记进行了测序，以便合成特异性引物。因此，对于每个测试的标记，都分离出了几个YAC克隆，包括一个携带位于Cf4/Cf9基因座周围富含亮氨酸重复序列的克隆家族。
