A RELIABLE METHOD FOR RANDOM MUTAGENESIS - THE GENERATION OF MUTANT LIBRARIES USING SPIKED OLIGODEOXYRIBONUCLEOTIDE PRIMERS
A RELIABLE METHOD FOR RANDOM MUTAGENESIS - THE GENERATION OF MUTANT LIBRARIES USING SPIKED OLIGODEOXYRIBONUCLEOTIDE PRIMERS
复制标题
DOI：
10.1016/0378-1119(89)90148-0
复制
发表时间：
1989-12-07
期刊：
影响因子：
3.5
通讯作者：
KNOWLES, JR
中科院分区：
文献类型：
--
作者：
HERMES, JD;PAREKH, SM;KNOWLES, JR
描述了一种用于生产确定的随机突变体文库的新方法。制备长掺入的寡脱氧核糖核苷酸（oligo）作为标准寡核苷酸定向诱变方案的引物，其中在每个位置使用预定水平的三个“错误"亚磷酰胺。在DNA合成仪上进行的加标寡核苷酸合成使用在线混合程序实现，该程序仅需要五个亚磷酰胺储存器，并且避免了任何纯亚磷酰胺试剂的污染。不可变的位置（即，可以指定寡核苷酸中使用纯试剂的位置），并且可以包括沉默的“标记"碱基，其允许诱变效率的早期估计。文库在改变的碱基的数量、类型和位置方面的随机性很容易通过DNA测序来验证。这个程序已被用来产生一个随机突变体库的基因编码一个缓慢的磷酸丙糖异构酶。在来自该文库的转化体中，已经发现许多第二位点抑制突变，其增加起始异构酶的比催化活性。该方法提供了比使用化学诱变试剂的方法更完整的文库。
A new procedure for the production of a defined library of random mutants is described. Long spiked oligodeoxyribonucleotides (oligos), in which a predetermined level of the three ''wrong'' phosphoramidites are used at each position, are made as primers for a standard oligo-directed mutagenesis protocol. Spiked oligo synthesis on a DNA synthesizer is achieved using an in-line mixing procedure that only requires five phosphoramidite reservoirs and which avoids contamination of any of the pure phosphoramidite reagents. Immutable positions (i.e., positions in the oligo for which pure reagents are used) can be specified, and a silent ''marker'' base can be included that allows an early estimate of the mutagenesis efficiency. The randomness of the library in respect to the number, type, and position of the altered bases, is easily verified by DNA sequencing. This procedure has been used to generate a random mutant library of the gene encoding a sluggish triosephosphate isomerase. Among the transformants from this library, a number of second-site suppressor mutations have been found that increase the specific catalytic activity of the starting isomerase. This approach provides a more complete library than a method using chemical mutagenic reagents.