Linear plasmid vector for cloning of repetitive or unstable sequences in Escherichia coli.
Linear plasmid vector for cloning of repetitive or unstable sequences in Escherichia coli.
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DOI：
10.1093/nar/gkp1181
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发表时间：
2010-04
影响因子：
14.9
通讯作者：
Ravin N
中科院分区：
文献类型：
--
作者：
Godiska R;Mead D;Dhodda V;Wu C;Hochstein R;Karsi A;Usdin K;Entezam A;Ravin N
尽管最近在测序方面取得了进展，但大基因组的完整完成和它们编码的新蛋白质的分析通常需要克隆特定区域。然而，这些片段中的许多片段极难在当前载体中克隆。环状质粒中的超螺旋应力可以产生作为缺失底物的二级结构，特别是在含有大量串联或反向重复序列的区域中。常见的载体也诱导插入片段的转录和翻译，其可以针对含有开放阅读框或重复DNA的重组克隆进行选择。相反，从克隆启动子的转录可干扰质粒稳定性。因此，我们开发了一种新的大肠杆菌克隆载体（称为“pJAZZ”载体），它是作为线性质粒。此外，它在克隆位点的两侧含有转录终止子，以使载体和插入物之间的转录干扰最小化。我们表明，该载体稳定地保持了各种插入，在传统的质粒是不可克隆的。这些靶标包括短的核苷酸重复序列，如扩增的脆性X基因座的那些，和大的富含AT的插入物，如来自肺孢子虫属、疟原虫属、尖毛虫属或四膜虫属的基因组DNA的20-kb片段。pJAZZ载体在克隆中显示出降低的大小偏差，允许在文库中更均匀地表示较大的片段。
Despite recent advances in sequencing, complete finishing of large genomes and analysis of novel proteins they encode typically require cloning of specific regions. However, many of these fragments are extremely difficult to clone in current vectors. Superhelical stress in circular plasmids can generate secondary structures that are substrates for deletion, particularly in regions that contain numerous tandem or inverted repeats. Common vectors also induce transcription and translation of inserted fragments, which can select against recombinant clones containing open reading frames or repetitive DNA. Conversely, transcription from cloned promoters can interfere with plasmid stability. We have therefore developed a novel Escherichia coli cloning vector (termed ‘pJAZZ’ vector) that is maintained as a linear plasmid. Further, it contains transcriptional terminators on both sides of the cloning site to minimize transcriptional interference between vector and insert. We show that this vector stably maintains a variety of inserts that were unclonable in conventional plasmids. These targets include short nucleotide repeats, such as those of the expanded Fragile X locus, and large AT—rich inserts, such as 20-kb segments of genomic DNA from Pneumocystis, Plasmodium, Oxytricha or Tetrahymena. The pJAZZ vector shows decreased size bias in cloning, allowing more uniform representation of larger fragments in libraries.
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影响因子：
12.3
作者：
Celniker SE;Wheeler DA;Kronmiller B;Carlson JW;Halpern A;Patel S;Adams M;Champe M;Dugan SP;Frise E;Hodgson A;George RA;Hoskins RA;Laverty T;Muzny DM;Nelson CR;Pacleb JM;Park S;Pfeiffer BD;Richards S;Sodergren EJ;Svirskas R;Tabor PE;Wan K;Stapleton M;Sutton GG;Venter C;Weinstock G;Scherer SE;Myers EW;Gibbs RA;Rubin GM
通讯作者：
Rubin GM
DOI：
10.1073/pnas.97.14.7721
发表时间：
2000-07-05
影响因子：
11.1
作者：
Deneke, J;Ziegelin, G;Lanka, E
通讯作者：
Lanka, E
影响因子：
7.7
作者：
Kouprina, N;Leem, SH;Larionov, V
通讯作者：
Larionov, V
影响因子：
7.7
作者：
Cui, Tailin;Moro-oka, Naoki;Horiuchi, Takashi
通讯作者：
Horiuchi, Takashi
影响因子：
2.9
作者：
Grabczyk, E;Usdin, K
通讯作者：
Usdin, K