Jenkins Glyn, Hasterok Robert
Institute of Biological Sciences, University of Wales Aberystwyth, Penglais, Aberystwyth, Ceredigion SY23 3DA, UK.
Nat Protoc. 2007;2(1):88-98. doi: 10.1038/nprot.2006.490.
Fluorescence in situ hybridization (FISH) using bacterial artificial chromosomes (BACs) with large genomic DNA inserts as probes (BAC 'landing') is a powerful means by which eukaryotic genomes can be physically mapped and compared. Here we report a BAC landing protocol that has been developed specifically for the weedy grass species Brachypodium distachyon, which has been adopted recently by the scientific community as an alternative model for the temperate cereals and grasses. The protocol describes the preparation of somatic and meiotic chromosome substrates for FISH, the labeling of BACs, a chromosome mapping strategy, empirical conditions for optimal in situ hybridization and stringency washing, the detection of probes and the capturing and processing of images. The expected outcome of the protocol is the specific assignment of BACs containing single-copy inserts to one of the five linkage groups of the genome of this species. Once somatic or meiotic material is available, the entire protocol can be completed in about 3 d. The protocol has been customized empirically for B. distachyon and its near relatives, but it can be adapted with minor modifications to diverse plant species.
使用带有大片段基因组DNA插入片段的细菌人工染色体（BAC）作为探针的荧光原位杂交（FISH）（BAC“着陆”法）是一种强大的手段，通过它可以对真核生物基因组进行物理图谱绘制和比较。在此，我们报告一种专门为杂草状禾本科植物二穗短柄草开发的BAC着陆方案，该植物最近被科学界采纳为温带谷类作物和禾本科植物的替代模式植物。该方案描述了用于FISH的体细胞和减数分裂染色体底物的制备、BAC的标记、染色体图谱绘制策略、最佳原位杂交和严谨性洗涤的经验条件、探针的检测以及图像的捕获和处理。该方案的预期结果是将包含单拷贝插入片段的BAC特异性定位到该物种基因组的五个连锁群之一。一旦获得体细胞或减数分裂材料，整个方案大约可在3天内完成。该方案是根据二穗短柄草及其近缘种的经验进行定制的，但稍作修改后可适用于多种植物物种。
