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

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

Avicenna J Med Biotechnol

Department of Biochemistry, Isfahan Pharmaceutical Sciences Research Center and Bioinformatics Research Center, School of Pharmacy, Isfahan University of Medical Sciences, Isfahan, Iran.

Published: January 2023

AI Article Synopsis

  • The study explores the effectiveness of Polyketide Synthase (PKS) and Nonribosomal Peptide Synthetase (NRPS) gene amplification to discover new therapeutic agents, while addressing issues caused by laboratory technique errors.
  • Using optimized PCR methods on 22 actinomycete strains, the researchers successfully amplified and sequenced several gene groups, revealing high frequencies of PKS and NRPS genes.
  • Results indicated that a significant portion of the strains contained important genes, and the study concluded that careful optimization of PCR conditions is crucial for accurate outputs and future therapeutic discoveries.

Article Abstract

Background: The screen of Polyketide Synthase () and Nonribosomal Peptide Synthetase () 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 and gene groups in previously isolated strains by optimizing their specialized amplification by degenerate primers and indicating the evolutionary relationships with reference strains.

Methods: , , and 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 , and 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 (86.4%), (81.8%), and (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 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 , , and gene groups.

Download full-text PDF

Source
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC9895980PMC
http://dx.doi.org/10.18502/ajmb.v15i1.11422DOI Listing

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