SYSTEMATIC SCREENING OF YEAST ARTIFICIAL-CHROMOSOME LIBRARIES BY USE OF THE POLYMERASE CHAIN-REACTION
SYSTEMATIC SCREENING OF YEAST ARTIFICIAL-CHROMOSOME LIBRARIES BY USE OF THE POLYMERASE CHAIN-REACTION
复制标题
DOI：
10.1073/pnas.87.3.1213
复制
发表时间：
1990-02-01
影响因子：
11.1
通讯作者：
OLSON, MV
中科院分区：
文献类型：
--
作者：
GREEN, ED;OLSON, MV
我们开发了一种基于聚合酶链反应（PCR）的筛选含有人类DNA的酵母人工染色体（YAC）克隆有序阵列的方法。该方法旨在确定阳性克隆在YAC文库中的位置，该文库作为单个克隆储存在96孔微量滴定板中。PCR的高灵敏度和特异性允许检测从1920个或更多个YAC克隆的库制备的DNA中的靶序列。基于PCR的筛选方案分两个连续阶段进行，这有效地将阳性克隆的位置限制在4个微量滴定板（384个克隆）中。每个阳性克隆的最终定位是通过常规的DNA·DNA杂交，使用含有来自适当的四个微量滴定板的YAC克隆的单个过滤器完成的。这种基于PCR的筛选策略已被证明是高效的，允许鉴定和分离许多含有特定人类基因的YAC克隆。发展战略的前景进行了讨论，完全基于PCR检测筛选YAC库，因为是这种方法的系统分析人类基因组的潜在应用。
We have developed an approach for screening ordered arrays of yeast artificial-chromosome (YAC) clones containing human DNA that is based on the polymerase chain reaction (PCR). This approach is designed to determined the locations of positive clones within a YAC library that is stored as individual clones in 96-well microtiter plates. The high sensitivity and specificity of the PCR allow the detection of target sequences in DNA prepared from pools of 1920 or more YAC clones. The PCR-based screening protocol is performed in two successive stages, which effectively limit the location of a positive clone to four microtiter plates (384 clones). Final localization of each positive clone is accomplished by conventional DNA.cntdot.DNA hybridization using a single filter containing the YAC clones from the appropriate four microtiter plates. This PCR-based screening strategy has proven highly efficient, allowing the identification and isolation of numerous YAC clones containing specific human genes. The prospects of developing a strategy for screening YAC libraries based completely on PCR assays are discussed, as are the potential applications of this approach to the systematic analysis of the human genome.