一种基于构件重组建立超大容量DNA文库的方法

Module shuffling for efficient combinatorial DNA library

设计固定长度的随机序列,并在随机序列的末端加上共用接头作为标准构件,利用共用接头之间的互补连接,用重聚PCR的方法将标准构件进行随机组合,得到1个序列多样化的DNA文库。重聚优化的条件为:随机序列质量浓度18 ng/μL,重聚PCR退火温度27.8℃,终止引物浓度为随机序列浓度的1/40。文中方法的建立克服了目前突变体文库建立方法中强烈依赖起始基因的弊端,可以开拓更为广阔的序列空间。

A mutant DNA library was formed with the design of fixed length random sequence,the addition of the same linker sequence to its ends,the sequences acting as standard module to hybridize and link each other in reassembly PCR process,and DNA sequences were randomly reassembled into full-length,chimeric sequences.The concentration of random sequence was 18 ng/μL and the concentration of termination primer 1/40 of that of random sequence.The anneal temperature of PCR was 27.8 ℃.Under the optimum conditions,the obtained DNA sequence with wide space solved problems of methods established in the generating mutant library relied on starting sequence.