Macrorestriction analysis of Caenorhabditis elegans genomic DNA.
Macrorestriction analysis of Caenorhabditis elegans genomic DNA.
复制标题
秀丽隐杆线虫基因组 DNA 的宏观限制分析。
DOI：
10.1093/genetics/144.2.609
复制
发表时间：
1996
期刊：
影响因子：
3.3
通讯作者：
Strome,S
中科院分区：
文献类型：
--
作者：
Browning,H;Berkowitz,L;Madej,C;Paulsen,JE;Zolan,ME;Strome,S
基因组物理图谱的有用性通过物理图谱与遗传图谱的连接而大大增强。我们描述了一个“macrorestriction映射”程序forCaenarhabditis elegans，我们已经应用到这一奋进。高分子量的基因组DNA用不常切割的限制性内切酶消化，并通过脉冲场凝胶电泳进行大小分级。用C.优美的物理地图这个过程允许构建覆盖几百个内切酶的限制性图谱，并使用定位几百个内切酶的探针检测多态性限制性片段。我们描述了这种技术的几个应用。(1)我们确定了先前未克隆区域中的DNA量<220 kb。(2)通过检测与该基因的缺失等位基因相关的多态性限制性片段，我们将themes-1基因定位到粘粒上。最初使用距离基因约400 kb的探针序列检测25-kb缺失。(3)我们绘制了缺陷hDf 6的分子终点图，并确定了unc-38-dpy-5区域的三个自发衍生的重复序列具有非常复杂的分子结构，包含内部重排和缺失。
The usefulness of genomic physical maps is greatly enhanced by linkage of the physical map with the genetic map. We describe a “macrorestriction mapping” procedure forCaenarhabditis elegansthat we have applied to this endeavor. High molecular weight, genomic DNA is digested with infrequently cutting restriction enzymes and size-fractionated by pulsed field gel electrophoresis. Southern blots of the gels are probed with clones from theC. elegansphysical map. This procedure allows the construction of restriction maps covering several hundred kilobases and the detection of polymorphic restriction fragments using probes that map several hundred kilobases away. We describe several applications of this technique. (1) We determined that the amount of DNA in a previously uncloned region is <220 kb. (2) We mapped themes-1gene to a cosmid, by detecting polymorphic restriction fragments associated with a deletion allele of the gene. The 25-kb deletion was initially detected using as a probe sequences located ~400 kb away from the gene. (3) We mapped the molecular endpoint of the deficiencyhDf6, and determined that three spontaneously derived duplications in theunc-38-dpy-5region have very complex molecular structures, containing internal rearrangements and deletions.