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Abstract <p>Researchers explore methods to enhance genetic material transfer into living organisms. While plasmids are commonly used to deliver genetic fragments for stable expression, they often suffer from inefficiencies, high costs, and time-consuming procedures. To address these challenges, we propose a novel cloning method that significantly reduces reliance on restriction enzymes. This method involves the separate amplification of each DNA strand using primers designed with specific sticky ends. The resulting single strands are then annealed to form a double-stranded product with compatible ends, facilitating seamless insertion into plasmids. To validate this method, plasmids containing microRNA-(21, 125, 486) precursors were successfully constructed and sequenced. These plasmids were then transfected into HEK293 and MCF7 cell lines to assess the expression levels of microRNAs in the cells. This approach not only proved to be cost-effective and time-efficient but also holds significant promise for a wide range of applications in genetics, biotechnology, and molecular biology. The method’s simplicity and efficiency make it a valuable tool for researchers aiming to streamline the cloning process and enhance the study of microRNA functions and gene expression.</p>

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Restriction enzyme-free method for generating PCR product sticky ends in recombinant vector construction for cloning

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2025年4月17日

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  • Mohammad Hashemabadi,
  • Mojdeh Amandadi,
  • Mojtaba Shaban,
  • Hosseinali Sasan

摘要

Abstract

Researchers explore methods to enhance genetic material transfer into living organisms. While plasmids are commonly used to deliver genetic fragments for stable expression, they often suffer from inefficiencies, high costs, and time-consuming procedures. To address these challenges, we propose a novel cloning method that significantly reduces reliance on restriction enzymes. This method involves the separate amplification of each DNA strand using primers designed with specific sticky ends. The resulting single strands are then annealed to form a double-stranded product with compatible ends, facilitating seamless insertion into plasmids. To validate this method, plasmids containing microRNA-(21, 125, 486) precursors were successfully constructed and sequenced. These plasmids were then transfected into HEK293 and MCF7 cell lines to assess the expression levels of microRNAs in the cells. This approach not only proved to be cost-effective and time-efficient but also holds significant promise for a wide range of applications in genetics, biotechnology, and molecular biology. The method’s simplicity and efficiency make it a valuable tool for researchers aiming to streamline the cloning process and enhance the study of microRNA functions and gene expression.