Site-specific genomic (SSG) and random domain-localized (RDL) mutagenesis in yeast.
Site-specific genomic (SSG) and random domain-localized (RDL) mutagenesis in yeast.
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DOI：
10.1186/1472-6750-4-7
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发表时间：
2004-04-16
影响因子：
3.5
通讯作者：
Honigberg SM
中科院分区：
文献类型：
--
作者：
Gray M;Kupiec M;Honigberg SM
酵母遗传学家可利用的一个有价值的武器是将特定突变引入酵母基因组的能力。特别是，已经开发了使用PCR片段将缺失引入酵母基因组的方法。这些方法效率很高，因为它们不需要在质粒中克隆。我们修改了现有的在酵母（酿酒酵母）基因组中引入缺失的方法，使用PCR片段来靶向该基因组的点突变。我们描述了两种基于pcr的方法，用于将点突变引导到酵母基因组中，从而使最终产物不含其他干扰。在第一种方法中，位点特异性基因组（SSG）诱变，一个特定的点突变被靶向到基因组中。在第二种方法中，随机区域定位（RDL）诱变，在基因的特定区域内随机引入突变。这两种方法都需要两次顺序转化，第一次转化将URA3标记整合到目标位点，第二次转化用包含一个或几个突变的PCR片段替换URA3。该PCR片段是用含有突变的引物合成的（SSG诱变）或通过容易出错的PCR合成的（RDL诱变）。在SSG突变中，URA3位点近端的突变比远端的突变合并的频率更高，然而突变可以在距离URA3插入至少500 bp的距离上有效引入。在RDL诱变中，为了确保突变在整个目标区域以大约相同的频率发生，在整合URA3的同时删除该区域。SSG和RDL诱变使点突变能够在不破坏原生位点的情况下轻松有效地整合到酵母基因组中。
A valuable weapon in the arsenal available to yeast geneticists is the ability to introduce specific mutations into yeast genome. In particular, methods have been developed to introduce deletions into the yeast genome using PCR fragments. These methods are highly efficient because they do not require cloning in plasmids. We have modified the existing method for introducing deletions in the yeast (S. cerevisiae) genome using PCR fragments in order to target point mutations to this genome. We describe two PCR-based methods for directing point mutations into the yeast genome such that the final product contains no other disruptions. In the first method, site-specific genomic (SSG) mutagenesis, a specific point mutation is targeted into the genome. In the second method, random domain-localized (RDL) mutagenesis, a mutation is introduced at random within a specific domain of a gene. Both methods require two sequential transformations, the first transformation integrates the URA3 marker into the targeted locus, and the second transformation replaces URA3 with a PCR fragment containing one or a few mutations. This PCR fragment is synthesized using a primer containing a mutation (SSG mutagenesis) or is synthesized by error-prone PCR (RDL mutagenesis). In SSG mutagenesis, mutations that are proximal to the URA3 site are incorporated at higher frequencies than distal mutations, however mutations can be introduced efficiently at distances of at least 500 bp from the URA3 insertion. In RDL mutagenesis, to ensure that incorporation of mutations occurs at approximately equal frequencies throughout the targeted region, this region is deleted at the same time URA3 is integrated. SSG and RDL mutagenesis allow point mutations to be easily and efficiently incorporated into the yeast genome without disrupting the native locus.
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影响因子：
3.9
作者：
Scott-Drew, S;Wong, CMVL;Murray, JAH
通讯作者：
Murray, JAH
影响因子：
2.1
作者：
Purnapatre, K;Piccirillo, S;Honigberg, SM
通讯作者：
Honigberg, SM
影响因子：
2.6
作者：
AMBERG, DC;BOTSTEIN, D;BEASLEY, EM
通讯作者：
BEASLEY, EM
影响因子：
5.3
作者：
Sagee, S;Sherman, A;Kassir, Y
通讯作者：
Kassir, Y
影响因子：
64.8
作者：
Giaever, G;Chu, AM;Johnston, M
通讯作者：
Johnston, M