Human BAC library: Construction and rapid screening
Human BAC library: Construction and rapid screening
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DOI：
10.1016/s0378-1119(97)00044-9
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发表时间：
1997-05-20
期刊：
影响因子：
3.5
通讯作者：
Shimizu, N
中科院分区：
文献类型：
--
作者：
Asakawa, S;Abe, I;Shimizu, N
我们利用正常雄性二倍体核型的前-亲- b细胞系FLEB14-14的高分子量DNA构建了人类基因组细菌人工染色体文库。该BAC文库包含96000个克隆，平均DNA插入长度为110 kb，覆盖人类基因组约3倍。库可以通过三种不同的方法进行筛选。(1)对31个高密度复制（HDR）过滤器进行探针杂交：每个过滤器包含3072个BAC克隆，以6 × 6的模式网格化。(2)将3072个BAC克隆的31个HindIII消化酶装载到两个Southern blot过滤器上进行探针杂交。这将确定一个特定的HDR过滤器，对其执行进一步的探针杂交以识别特定的克隆。(3)两步聚合酶链式反应（PCR）。首先，将PCR应用于从9600个BAC克隆的10个超级池中制备的DNA样本，以确定一个特定的超级池；第二次PCR应用于从特定超级池的四维指定BAC克隆制备的40个独特的DNA样本。本文给出了使用这三种方法进行文库筛选的典型例子。两步PCR筛选特别强大，因为它使我们能够在一天左右的时间内分离出所需的BAC克隆。对该方法的优越性进行了理论分析。此外，我们已将Vectorette方法应用于我们的BAC文库，用于分离BAC DNA插入的末端序列，以促进BAC行走的构象形成。(C) 1997爱思唯尔科学有限公司
We have constructed a human genomic bacterial artificial chromosome (BAG) library using high molecular weight DNA from a pre-pro-B cell line, FLEB14-14, with a normal male diploid karyotype. This BAC library consists of 96 000 clones with an average DNA insert size of 110 kb, covering the human genome approximately 3 times. The library can be screened by three different methods. (1) Probe hybridization to 31 high-density replica (HDR) filters: each filter contains 3072 BAC clones which were gridded in a 6 x 6 pattern. (2) Probe hybridization to two Southern blot filters to which 31 HindIII digests of the pooled 3072 BAC clones were loaded. This identifies a particular HDR filter for which further probe hybridization is performed to identify a particular clone(s). (3) Two-step polymerase chain reaction (PCR). First, PCR is applied to DNA samples prepared from ten superpools of 9600 BAC clones each to identify a particular superpool and the second PCR is applied to 40 unique DNA samples prepared from the four-dimensionally assigned BAC clones of the particular superpool. We present typical examples of the library screening using these three methods. The two-step PCR screening is particularly powerful since it allows us to isolate a desired BAC clone(s) within a day or so. The theoretical consideration of the advantage of this method is presented. Furthermore, we have adapted Vectorette method to our BAC library for the isolation of terminal sequences of the BAC DNA insert to facilitate contig formation by BAC walking. (C) 1997 Elsevier Science B.V.