Synthesis of libraries and multi-site mutagenesis using a PCR-derived, dU-containing template.
Synthesis of libraries and multi-site mutagenesis using a PCR-derived, dU-containing template.
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DOI：
10.1093/synbio/ysaa030
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发表时间：
2021
期刊：
影响因子：
--
通讯作者：
Bohm A
中科院分区：
文献类型：
--
作者：
Meinke G;Dalda N;Brigham BS;Bohm A
定向DNA文库很有用，因为它们集中在序列中最重要的区域的遗传多样性。理想情况下，这样的文库中的所有序列应该以相同的频率出现，并且不应该有来自起始序列的显著背景。这些属性最大化了可以筛选的不同序列的数量。本文描述了一种称为SLUPT(通过含DU的PCR衍生模板合成文库)的方法，用于产生高靶向DNA文库和/或多点突变，其中改变的碱基可以广泛分布在靶序列中。该方法具有高效、模块化的特点。此外，可以在一次反应中改变多个不同的位置，每个位置都有一个或多个碱基变化。起始序列有非常低的背景，并且SLUPT文库在选择变异的位置具有每个碱基的相似表示。SLUPT方法利用由聚合酶链式反应(PCR)制成的含有Du的单链DNA模板。以这种方式合成模板比前面描述的要容易得多。一系列与模板同源并编码所需遗传多样性的寡核苷酸引物在单个反应中延伸和连接，以形成突变产物序列或文库。模板选择性失活后，仅扩增产物文库。除了它们不能重叠外，对诱变引物的间距没有限制。
Directed DNA libraries are useful because they focus genetic diversity in the most important regions within a sequence. Ideally, all sequences in such libraries should appear with the same frequency and there should be no significant background from the starting sequence. These properties maximize the number of different sequences that can be screened. Described herein is a method termed SLUPT (Synthesis of Libraries via a dU-containing PCR-derived Template) for generating highly targeted DNA libraries and/or multi-site mutations wherein the altered bases may be widely distributed within a target sequence. This method is highly efficient and modular. Moreover, multiple distinct sites, each with one or more base changes, can be altered in a single reaction. There is very low background from the starting sequence, and SLUPT libraries have similar representation of each base at the positions selected for variation. The SLUPT method utilizes a single-stranded dU-containing DNA template that is made by polymerase chain reaction (PCR). Synthesis of the template in this way is significantly easier than has been described earlier. A series of oligonucleotide primers that are homologous to the template and encode the desired genetic diversity are extended and ligated in a single reaction to form the mutated product sequence or library. After selective inactivation of the template, only the product library is amplified. There are no restrictions on the spacing of the mutagenic primers except that they cannot overlap.
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DOI：
10.1056/nejmoa1504030
发表时间：
2015-07-02
期刊：
The New England journal of medicine
影响因子：
--
作者：
Larkin J;Chiarion-Sileni V;Gonzalez R;Grob JJ;Cowey CL;Lao CD;Schadendorf D;Dummer R;Smylie M;Rutkowski P;Ferrucci PF;Hill A;Wagstaff J;Carlino MS;Haanen JB;Maio M;Marquez-Rodas I;McArthur GA;Ascierto PA;Long GV;Callahan MK;Postow MA;Grossmann K;Sznol M;Dreno B;Bastholt L;Yang A;Rollin LM;Horak C;Hodi FS;Wolchok JD
通讯作者：
Wolchok JD
影响因子：
48
作者：
Gibson, Daniel G.;Young, Lei;Smith, Hamilton O.
通讯作者：
Smith, Hamilton O.
影响因子：
4.7
作者：
Chaparro-Riggers, Javier F.;Polizzi, Karen M.;Bommarius, Andreas S.
通讯作者：
Bommarius, Andreas S.
治疗性 ipilimumab 和 tremelimumab 抗体的 CTLA-4 结合特性非常相似
影响因子：
--
作者：
He M;Chai Y;Qi J;Zhang CWH;Tong Z;Shi Y;Yan J;Tan S;Gao GF
通讯作者：
Gao GF
影响因子：
64.8
作者：
CLACKSON, T;HOOGENBOOM, HR;WINTER, G
通讯作者：
WINTER, G