A simple, flexible and efficient PCR-fusion/Gateway cloning procedure for gene fusion, site-directed mutagenesis, short sequence insertion and domain deletions and swaps.
A simple, flexible and efficient PCR-fusion/Gateway cloning procedure for gene fusion, site-directed mutagenesis, short sequence insertion and domain deletions and swaps.
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DOI：
10.1186/1746-4811-5-14
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发表时间：
2009-10-28
期刊：
影响因子：
5.1
通讯作者：
Turner SR
中科院分区：
文献类型：
--
作者：
Atanassov II;Atanassov II;Etchells JP;Turner SR
各种植物基因组测序计划的进展和完成为涉及克隆、修饰和随后表达目的基因的各种功能基因组研究铺平了道路。这需要用于产生二元载体的灵活且有效的程序，所述二元载体包含：基因融合体、来自定点诱变的变体、添加蛋白质标签以及结构域交换和缺失。此外，有效的克隆程序，理想的高通量，是必不可少的多基因构建体的克隆。在这里，我们提出了一个简单，灵活和有效的PCR融合/网关克隆程序的二元载体的建设范围内的基因融合或变异与单或多个核苷酸取代，短序列插入，结构域缺失和交换。展示了该程序的选定应用的结果，包括ORF融合、Cys>Ser突变的引入、StrepII标签序列的插入和拟南芥次生细胞壁AtCesA基因的结构域交换。所描述的PCR融合/网关克隆程序为广泛多样和复杂的克隆任务提供了优雅、简单和有效的解决方案。通过将基因融合体和修饰变体的集合精简克隆到二元载体中用于基因家族的系统功能研究，我们的方法允许有效利用不断增长的序列和表达数据。
The progress and completion of various plant genome sequencing projects has paved the way for diverse functional genomic studies that involve cloning, modification and subsequent expression of target genes. This requires flexible and efficient procedures for generating binary vectors containing: gene fusions, variants from site-directed mutagenesis, addition of protein tags together with domain swaps and deletions. Furthermore, efficient cloning procedures, ideally high throughput, are essential for pyramiding of multiple gene constructs. Here, we present a simple, flexible and efficient PCR-fusion/Gateway cloning procedure for construction of binary vectors for a range of gene fusions or variants with single or multiple nucleotide substitutions, short sequence insertions, domain deletions and swaps. Results from selected applications of the procedure which include ORF fusion, introduction of Cys>Ser mutations, insertion of StrepII tag sequence and domain swaps for Arabidopsis secondary cell wall AtCesA genes are demonstrated. The PCR-fusion/Gateway cloning procedure described provides an elegant, simple and efficient solution for a wide range of diverse and complicated cloning tasks. Through streamlined cloning of sets of gene fusions and modification variants into binary vectors for systematic functional studies of gene families, our method allows for efficient utilization of the growing sequence and expression data.
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影响因子：
9.2
作者：
Liu, QH;Li, MZ;Elledge, SJ
通讯作者：
Elledge, SJ
影响因子：
1.6
作者：
Nakagawa, Tsuyoshi;Suzuki, Takarnasa;Ishiguro, Sumie
通讯作者：
Ishiguro, Sumie
影响因子：
7.4
作者：
Dafny-Yelin, Mery;Tzfira, Tzvi
通讯作者：
Tzfira, Tzvi
影响因子：
4.1
作者：
Sasaki, Y;Sone, T;Imamoto, F
通讯作者：
Imamoto, F
影响因子：
3.5
作者：
INOUE, H;NOJIMA, H;OKAYAMA, H
通讯作者：
OKAYAMA, H