A genetic system for direct selection of gene-positive clones during recombinational cloning in yeast
A genetic system for direct selection of gene-positive clones during recombinational cloning in yeast
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DOI：
10.1093/nar/30.2.e8
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发表时间：
2002-01-15
影响因子：
14.9
通讯作者：
Larionov, V
中科院分区：
文献类型：
--
作者：
Noskov, V;Kouprina, N;Larionov, V
转化相关重组(TAR)是一种无需预先构建基因组文库而直接从基因组DNA中分离特定染色体区域或基因的克隆技术。这项技术涉及基因组DNA和具有5‘和3’基因靶向序列(挂钩)的TAR载体之间的球体转化过程中的同源重组。通常，TAR克隆产生阳性YAC重组子的频率类似于0.5%；阳性克隆通过聚合酶链式反应或菌落杂交进行鉴定。本文描述了一种新的焦油克隆方法，即通过正、负遗传选择来选择正克隆。该系统使用带有两个靶向挂钩的TAR载体，HISS作为正选择标记，URA3作为负选择标记和称为环序列的基因特异性序列。该环序列位于染色体靶中的靶向挂钩序列的远端，但在TAR载体中的靶向挂钩和URA3的近端。当该载体在基因特异性靶向钩处与染色体DNA重组时，重组YAC产物携带两个副本的环序列，因此，URA3负选择标记在有丝分裂中变得不稳定，并通过涉及环序列的直接重复重组而在高频下丢失。通过对URA3进行选择来鉴定阳性克隆。这种方法产生阳性YAC重组子的频率接近40%。这种新的TAR克隆方法为复杂基因组的结构和功能分析提供了强有力的工具。
Transformation-associated recombination (TAR) Is a cloning technique that allows specific chromosomal regions or genes to be isolated directly from genomic DNA without prior construction of a genomic library. This technique Involves homologous recombination during spheroplast transformation between genomic DNA and a. TAR vector that has 5' and 3' gene targeting sequences (hooks). Typically, TAR cloning produces positive YAC recombinants at a frequency of similar to0.5%; the positive clones are identified by PCR or colony hybridization. This paper describes a novel TAR cloning procedure that selects positive clones by positive and negative genetic selection. This system utilizes a TAR vector with two targeting hooks, HISS as a positive selectable marker, URA3 as a negative selectable marker and a gene-specific sequence called a loop sequence. The loop sequence lies distal to a targeting hook sequence in the chromosomal target, but proximal to the targeting hook and URA3 in the TAR vector. When this vector recombines with chromosomal DNA at the gene-specific targeting hook, the recombinant YAC product carries two copies of the loop sequence, therefore, the URA3 negative selectable marker becomes mitotically unstable and is, lost at high frequency by direct repeat recombination Involving the loop sequence. Positive clones are identified by selecting against URA3. This method produces positive YAC recombinants at a frequency of similar to40%. This novel TAR cloning method provides a powerful tool for structural and functional analysis of complex genomes.