Optimization of Degenerate PCR Conditions for Reducing Error Rates in Detection of PKS and NRPS Gene groups in Actinomycetes

Sara  Ghashghaei; Zahra  Etemadifar; Manoochehr  Tavassoli; Mohammad Reza  Mofid

doi:10.18502/ajmb.v15i1.11422

Sara Ghashghaei Department of Cell and Molecular Biology & Microbiology, Faculty of Biological Science and Technology, University of Isfa-han, Isfahan, Iran
Zahra Etemadifar Department of Cell and Molecular Biology & Microbiology, Faculty of Biological Science and Technology, University of Isfa-han, Isfahan, Iran
Manoochehr Tavassoli Department of Cell and Molecular Biology & Microbiology, Faculty of Biological Science and Technology, University of Isfa-han, Isfahan, Iran
Mohammad Reza Mofid Department of Biochemistry, Isfahan Pharmaceutical Sciences Research Center and Bioinformatics Research Center, School of Pharmacy, Isfahan University of Medical Sciences, Isfahan, Iran

DOI: https://doi.org/10.18502/ajmb.v15i1.11422

Keywords: Antimicrobial peptides, Biological products, PCR, Polyketide synthase, Streptomyces

Abstract

Background: The screen of Polyketide Synthase (PKS) and Nonribosomal Peptide Synthetase (NRPS) gene groups is a quick way to discover new therapeutic agents. However, errors in laboratory techniques cause a loss of touch with reality. This study aimed to evaluate the presence of PKS and NRPS gene groups in previously isolated strains by optimizing their specialized amplification by degenerate primers and indicating the evolutionary relationships with reference strains.

Methods: PKS-I, II, and NRPS genes PCR amplification was performed using three degenerate primer sets for 22 actinomycete strains with antibacterial activity. Annealing temperature and the amount of template DNA and primers were optimized. PCR products of PKS-I, II, and NRPS from three strains were sequenced after TA cloning. Besides, strains with high antibacterial activity were identified by biochemical features and partial 16S rDNA sequencing and hypothetically classified by a phylogenetic tree.

Results: High frequencies of PKS-I (86.4%), PKS-II (81.8%), and NRPS (95.4%) genes were found among the strains after optimization. Fourteen strains (64%) contained all of the genes, and 100% of strains had at least two genes. These numbers are pretty distinct in comparison with the previous researches. All of the sequenced strains were members of Streptomyces genus.

Conclusion: Our research showed that degenerate PCR strongly depends on the variation of annealing temperature and primer concentration, resulting in an unexpected shift in PCR outputs. The sequencing results confirmed the optimized conditions for specialized PCR of PKS-I, PKS-II, and NRPS gene groups.