A high-throughput genome-walking method and its use for cloning unknown flanking sequences
A high-throughput genome-walking method and its use for cloning unknown flanking sequences
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DOI：
10.1016/j.ab.2008.07.012
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发表时间：
2008-10-15
影响因子：
2.9
通讯作者：
Reddy, Malireddy K.
中科院分区：
文献类型：
--
作者：
Reddy, Palakolanu Sudhakar;Mahanty, Srikrishna;Reddy, Malireddy K.
我们开发了一种基于PCR的高通量基因组步行协议。该方案的新奇在于，在将部分简并引物退火至变性的基因组DNA之后，通过利用Phi 29 DNA聚合酶的DNA合成的滚环模式，将独特的步行者引物结合位点有效地随机引入基因组的不同区域。Phi 29 DNA聚合酶的固有链置换活性置换下游链的5'端，DNA合成继续，导致大量重叠片段，其覆盖整个基因组，其中独特的步行者衔接子连接到所有基因组DNA片段的5'端。可以使用基因座特异性引物和步行者引物以及Phi 29 DNA聚合酶扩增的基因组DNA片段作为模板进行定向基因组步行。基因座特异性引物将决定基因组步移的位置和方向。通过基因座特异性引物和步行者引物及其相应的巢式引物进行两轮连续PCR扩增，有效地扩增了侧翼DNA片段。所需的PCR片段可以克隆或直接测序使用另一个巢式，基因座特异性引物。我们成功地使用该方案来分离和测序所选植物基因的5'侧翼区/启动子。(C)2008年爱思唯尔公司All rights reserved.
We developed a PCR-based high-throughput genome-walking Protocol. The novelty of this protocol is in the random introduction of unique walker primer binding sites into different regions of the genomes efficiently by taking advantage of the rolling circle mode of DNA synthesis by Phi29 DNA polymerase after annealing the partially degenerate primers to the denatured genomic DNA. The inherent strand-displacement activity of the Phi29 DNA polymerase displaces the 5' ends of downstream strands and DNA synthesis continues, resulting in a large number of overlapping fragments that cover the whole genome with the unique walker adapter attached to the 5' end of all the genomic DNA fragments. The directional genome walking can be performed using a locus-specific primer and the walker primer and Phi29 DNA polymerase-amplified genomic DNA fragments as template. The locus-specific primer will determine the position and direction of the genome walk. Two rounds of successive PCR amplifications by locus-specific and walker primers and their corresponding nested primers effectively amplify the flanking DNA fragments. The desired PCR fragment can be either cloned or sequenced directly Using another nested, locus-specific primer. We successfully used this protocol to isolate and sequence 5' flanking regions/promoters of selected plant genes. (C) 2008 Elsevier Inc. All rights reserved.