MutS-mediated enrichment of mutated DNA produced by directed evolution in vitro

Abstract

Directed evolution in vitro is a powerful tool in the study and design of protein function. However, screening the desired mutants is a difficult task. To facilitate the screening, a method is proposed to eliminate wild type sequences and increase mutated DNA sequences, which is based on the preferential binding of MutS protein to heteroduplex DNA. Following error-prone PCR, amplified products are denatured and re-annealed to form heteroduplex and homoduplex DNA. Heteroduplexes are selectively bound to an engineered MutS protein and immobilized on a Strep-Tactin column. Homoduplexes are effectively removed by washing, and the final elution is enriched in mutated DNA sequences. One round of mutated DNA enrichment resulted in an about 2.3-fold of increase in mutation frequency compared to the control. The percentage of mutants rose from 44% in the control sample to 72% in the enrichment sample. Fluorescent assay by flow cytometry showed that the enrichment method increased the mutants with changed fluorescent activity by about 2.2-fold, which strongly justified the efficiency of enrichment in increasing mutants with functional changes. With reduced workload of screening and increased possibility of obtaining mutants with functional changes, the overall efficiency was improved by MutS-mediated enrichment of mutated DNA.

Serial enrichment of heteroduplex DNA using a MutS‐magnetic bead system

“…[ 10 ] In comparison, we show 39.5‐fold enrichment of 1% heteroduplex DNA and enrichment of 5% heteroduplex DNA input to 100% heteroduplex DNA. The main difference is that prior methods conducted only one or two MutS enrichment cycles, [ 10,16–18 ] whereas our system has been optimized to enable a large number of serial enrichment cycles. Only two prior methods have achieved higher sensitivities and enrichment than our method, but they accomplished this by coupling MutS enrichment with subsequent allele‐specific PCR amplification.…”

“…Using these principles, bead-based, column-based, and resinbased MutS methods have been previously developed to enrich mutant DNA. [10,[16][17][18] While these methods can successfully enrich (or deplete) mutant DNA, they perform only one or two cycles of enrichment with MutS, which limits the degree of mutant DNA enrichment. [10,[16][17][18] Additionally, MutS has been combined with allele-specific PCR amplification to achieve mutant DNA enrichment.…”

“…[10,[16][17][18] While these methods can successfully enrich (or deplete) mutant DNA, they perform only one or two cycles of enrichment with MutS, which limits the degree of mutant DNA enrichment. [10,[16][17][18] Additionally, MutS has been combined with allele-specific PCR amplification to achieve mutant DNA enrichment. [19,20] We hypothesized that a method utilizing a larger number of serial MutS enrichment cycles could achieve even higher levels of heteroduplex DNA (and therefore mutant DNA) enrichment, without additional steps such as allele-specific PCR amplification.…”