Digital mapping of bacterial artificial chromosomes by fluorescence in situ hybridization
Digital mapping of bacterial artificial chromosomes by fluorescence in situ hybridization
复制标题
DOI：
10.1046/j.1365-313x.1999.00398.x
复制
发表时间：
1999-03-01
期刊：
影响因子：
7.2
通讯作者：
Jiang, JM
中科院分区：
文献类型：
--
作者：
Jackson, SA;Dong, FG;Jiang, JM
细菌人工染色体(BAC)已成为克隆大片段DNA最常用的工具。大多数BAC克隆的插入片段平均为100-200kb，而如此大的DNA片段的分子特征是一个主要的挑战。在这里，我们报告了一种简单方便的BAC插入物物理标测技术。将单个BAc分子固定在涂有聚L赖氨酸的玻片上。以BAC DNA为探针，用荧光原位杂交技术显示完整的环状BAC分子。7.4kb的BAC载体扩展到每微米约2.44kb。使用BAC载体的扩展作为标准校准，可以将数字测量的线性距离转换为千碱基的DNA。我们将小到2kb的DNA片段直接定位在环状BAC分子上。利用该技术对同时含有串联重复和分散重复的水稻BAC克隆进行了分析。有效地确定了不同重复序列在BAC插入体内的分布和组织。结果表明，该技术对于鉴定含有复杂重复DNA序列的BAC克隆特别有价值。
The bacterial artificial chromosome (BAC) has become the most popular tool for cloning large DNA fragments. The inserts of most BAC clones average 100-200 kilobases (kb) and molecular characterization of such large DNA fragments is a major challenge. Here we report a simple and expedient technique for physical mapping of BAC inserts. Individual BAC molecules were immobilized on glass slides coated with Poly-L-lysine. The intact circular BAC molecules were visualized by fluorescence in situ hybridization using BAC DNA as a probe. The 7.4 kb BAC vector was extended to approximately 2.44 kb per micrometer. Digitally measured linear distances can be transformed into kilobases of DNA using the extension of BAC vector as a standard calibration. We mapped DNA fragments as small as 2 kb directly on circular BAC molecules. A rice BAC clone containing both tandem and dispersed repeats was analyzed using this technique. The distribution and organization of the different repeats within the BAC insert were efficiently determined. The results showed that this technique will be especially valuable for characterizing BAC clones that contain complex repetitive DNA sequences.