Efficient PCR walking on plant genomic DNA
Efficient PCR walking on plant genomic DNA
复制标题
植物基因组 DNA 上的高效 PCR 行走
DOI：
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发表时间：
1997
影响因子：
6.5
通讯作者：
T. Roscoe
中科院分区：
文献类型：
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作者：
M. Devic;S. Albert;M. Delseny;T. Roscoe
我们将聚合酶链反应（PCR）在未克隆的人类基因组DNA上行走的技术应用于植物基因组DNA。在本文中，我们证明了PCR行走是一种高效、快速、简单和可重复的方法，用于鉴定由整合分子标记的植物基因和从植物DNA中分离调控序列。PCR行走法用于扩增转化系T-DNA左右边缘的长拟南芥基因组DNA序列。利用所得到的序列信息，利用不同长度和Tm的引物组合在野生型基因组DNA上行走，以表征T-DNA靶位点并克隆相应的野生型基因。我们证明了一种小规模、快速的分离方案提供了足够完整性的基因组DNA，使PCR行走适用于大量植物的常规分析。为了分离和表征拟南芥FAEI基因同源的开放阅读框上游的调控序列，我们在甘蓝型油菜中使用了这种方法。因此，DNA在植物基因组DNA上行走对于T-DNA侧翼区域的表征是一种非常有效的替代方法，并且消除了对基因组区域侧翼编码序列，ESTs， sts或任何短的已知序列的筛选文库的要求。
We have adapted the technique of polymerase chain reaction (PCR) walking on uncloned human genomic DNA to plant genomic DNA. In this paper we show that PCR walking is an efficient and rapid, yet simple and reproducible approach for the characterisation of the plant genes tagged by an integrative molecule and for the isolation of regulatory sequences from plant DNA. PCR walking was used to amplify long Arabidopsis genomic DNA sequences at the right and left borders of the T-DNA of the transformed lines. The resulting sequence information was used to walk with different primer combinations varying in length and in Tm on wild type genomic DNA in order to characterise the T-DNA target site and to clone the corresponding wild type gene. We demonstrate that a small scale, rapid isolation protocol provides genomic DNA of sufficient integrity to make PCR walking appropriate for the routine analysis of large number of plants. We have used this approach with Brassica napus in order to isolate and characterise the regulatory sequences upstream of an open reading frame homologous to the FAEI gene of Arabidopsis. Thus, DNA walking on plant genomic DNA is an extremely efficient alternative to inverse PCR for the characterization of T-DNA flanking regions and eliminates the requirement of screening libraries for the isolation of genomic regions flanking coding sequences, ESTs, STSs or any short, known sequence.