Weier H U
Department of Subcellular Structure, Life Sciences Division, University of California, Ernest Orlando Lawrence Berkeley National Laboratory, Berkeley, California, USA.
J Histochem Cytochem. 2001 Aug;49(8):939-48. doi: 10.1177/002215540104900802.
High-resolution physical maps are indispensable for directed sequencing projects or the finishing stages of shotgun sequencing projects. These maps are also critical for the positional cloning of disease genes and genetic elements that regulate gene expression. Typically, physical maps are based on ordered sets of large insert DNA clones from cosmid, P1/PAC/BAC, or yeast artificial chromosome (YAC) libraries. Recent technical developments provide detailed information about overlaps or gaps between clones and precisely locate the position of sequence tagged sites or expressed sequences, and thus support efforts to determine the complete sequence of the human genome and model organisms. Assembly of physical maps is greatly facilitated by hybridization of non-isotopically labeled DNA probes onto DNA molecules that were released from interphase cell nuclei or recombinant DNA clones, stretched to some extent and then immobilized on a solid support. The bound DNA, collectively called "DNA fibers," may consist of single DNA molecules in some experiments or bundles of chromatin fibers in others. Once released from the interphase nuclei, the DNA fibers become more accessible to probes and detection reagents. Hybridization efficiency is therefore increased, allowing the detection of DNA targets as small as a few hundred base pairs. This review summarizes different approaches to DNA fiber mapping and discusses the detection sensitivity and mapping accuracy as well as recent achievements in mapping expressed sequence tags and DNA replication sites.
高分辨率物理图谱对于定向测序项目或鸟枪法测序项目的完成阶段而言不可或缺。这些图谱对于疾病基因以及调控基因表达的遗传元件的定位克隆也至关重要。通常，物理图谱基于来自黏粒、P1/PAC/BAC或酵母人工染色体（YAC）文库的大插入片段DNA克隆的有序集合。近期的技术发展提供了有关克隆之间重叠或缺口的详细信息，并精确确定了序列标签位点或表达序列的位置，从而支持了确定人类基因组和模式生物完整序列的工作。通过将非同位素标记的DNA探针与从间期细胞核或重组DNA克隆中释放出来的DNA分子杂交，在一定程度上拉伸后固定在固体支持物上，极大地促进了物理图谱的组装。在某些实验中，结合的DNA，统称为“DNA纤维”，可能由单个DNA分子组成，而在其他实验中则可能由染色质纤维束组成。一旦从间期核中释放出来，DNA纤维就更容易被探针和检测试剂接触到。因此，杂交效率得以提高，能够检测到小至几百个碱基对的DNA靶标。本综述总结了DNA纤维图谱绘制的不同方法，并讨论了检测灵敏度和图谱绘制准确性，以及在绘制表达序列标签和DNA复制位点方面的最新成果。
