Tianying Zhong

Publications (3)9.41 Total impact

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

  Tianying Zhong, Yafeng Zhou, Lijun Bi, Xian-En Zhang
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  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. KeywordsDirected evolution–MutS–Mutated DNA enrichment–Mutation frequency
  World Journal of Microbiology and Biotechnology 04/2012; 27(6):1367-1372. · 1.53 Impact Factor
• Source

  Available from: library.ibp.ac.cn

  Article: New functional sites in MutS affect DNA mismatch repair.

  Tianying Zhong, Lijun Bi, Xianen Zhang
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  ABSTRACT: The MutS protein plays an important role in the DNA mismatch repair system. Mutations in the mutS gene can lead to genome instability and ultimately cell malfunction. Here we have established a method for identifying functional defective mutants of MutS by random mutation and rifampicin screening. Some novel functional sites in MutS were identified. The MutS mutant strains were analyzed using surface plasmon resonance, gel filtration and far-western methods to determine the molecular mechanisms behind the DNA mismatch repair function of MutS.
  Science China. Life sciences 10/2010; 53(10):1170-3. · 2.02 Impact Factor
• Source

  Available from: Bernie Kraatz

  Article: Unlabeled hairpin DNA probe for electrochemical detection of single-nucleotide mismatches based on MutS-DNA interactions.

  He Gong, Tianying Zhong, Lan Gao, Xiaohong Li, Lijun Bi, Heinz-Bernhard Kraatz
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  ABSTRACT: The paper described a label-free assay for the detection of single-nucleotide mismatches in which an unlabeled hairpin DNA probe and a MutS protein conjugate (His6-MutS-linker peptide-streptavidin binding peptide (HMLS)) are exploited for the detection of mismatches by electrochemical impedance spectroscopy (EIS). We demonstrate this method for eight single-nucleotide mismatches. Upon hybridization of the target strand with the hairpin DNA probe, the stem-loop structure is opened forming a duplex DNA. In duplexes containing a single nucleotide mismatch, the mismatch is present at the solvent exposed side, enabling more effective HMLS recognition and binding. The binding event is evaluated by EIS and analyzed with the help of Randles' equivalent circuits. The differences in the charge transfer resistance DeltaR(CT) before and after protein binding to the duplex DNA allows the unequivocal detection of all eight single-nucleotide mismatches. DeltaR(CT) allows the discrimination of a C-A mismatch with the concentration of the target strand as low as 100 pM.
  Analytical Chemistry 09/2009; 81(20):8639-43. · 5.86 Impact Factor