Compartmentalized self-replication under fast PCR cycling conditions yields Taq DNA polymerase mutants with increased DNA-binding affinity and blood resistance.
Compartmentalized self-replication under fast PCR cycling conditions yields Taq DNA polymerase mutants with increased DNA-binding affinity and blood resistance.
复制标题
DOI：
10.3389/fmicb.2014.00408
复制
发表时间：
2014
影响因子：
5.2
通讯作者：
Hogrefe H
中科院分区：
文献类型：
--
作者：
Arezi B;McKinney N;Hansen C;Cayouette M;Fox J;Chen K;Lapira J;Hamilton S;Hogrefe H
已经开发了更快循环的PCR制剂、方案和仪器，以满足基于PCR的测定对增加通量和缩短周转时间的需求。虽然运行时间可以缩短高达50%，但较短的循环时间与较低的检测灵敏度和增加的可变性相关。为了解决这些问题，我们应用区室化自我复制（CSR）来进化Taq DNA聚合酶的快速循环突变体。在使用逐渐缩短的PCR延伸时间进行五轮选择后，使用基于连接的多位点诱变随机组合在最快循环克隆中鉴定的单个突变。表现最好的组合突变体表现出35- 90倍高的亲和力（较低的Kd）引发的模板和适度（2倍）增加的延伸率相比，野生型Taq。进一步的表征显示，CSR选择的突变提供了对抑制剂的增加的抗性，并且最值得注意的是，能够从高达65%的全血中直接扩增。我们讨论了单个突变对快速循环和血液抗性表型的贡献。
Faster-cycling PCR formulations, protocols, and instruments have been developed to address the need for increased throughput and shorter turn-around times for PCR-based assays. Although run times can be cut by up to 50%, shorter cycle times have been correlated with lower detection sensitivity and increased variability. To address these concerns, we applied Compartmentalized Self Replication (CSR) to evolve faster-cycling mutants of Taq DNA polymerase. After five rounds of selection using progressively shorter PCR extension times, individual mutations identified in the fastest-cycling clones were randomly combined using ligation-based multi-site mutagenesis. The best-performing combinatorial mutants exhibit 35- to 90-fold higher affinity (lower Kd) for primed template and a moderate (2-fold) increase in extension rate compared to wild-type Taq. Further characterization revealed that CSR-selected mutations provide increased resistance to inhibitors, and most notably, enable direct amplification from up to 65% whole blood. We discuss the contribution of individual mutations to fast-cycling and blood-resistant phenotypes.
登录
查看更多内容
影响因子：
14.9
作者：
Wang, Y;Prosen, DE;Vander Horn, PB
通讯作者：
Vander Horn, PB
DOI：
10.1073/pnas.88.16.7276
发表时间：
1991-08-01
影响因子：
11.1
作者：
HOLLAND, PM;ABRAMSON, RD;GELFAND, DH
通讯作者：
GELFAND, DH
影响因子：
2.8
作者：
Watson, RJ;Blackwell, B
通讯作者：
Blackwell, B
聚合酶的分子育种，用于扩增古代DNA。
影响因子：
46.9
作者：
d'Abbadie, Marc;Hofreiter, Michael;Vaisman, Alexandra;Loakes, David;Gasparutto, Didier;Cadet, Jean;Woodgate, Roger;Paeaebo, Svante;Holliger, Philipp
通讯作者：
Holliger, Philipp
DOI：
10.1073/pnas.071052198
发表时间：
2001-04-10
影响因子：
11.1
作者：
Ghadessy, FJ;Ong, JL;Holliger, P
通讯作者：
Holliger, P