Application of PFU enzyme in genetic engineering

一、High – fidelity DNA Amplification

1. Gene Cloning
   • During the gene cloning process, it is necessary to accurately amplify the target gene from complex genomic DNA. Due to its high – fidelity property, the PFU enzyme can ensure that the amplified gene sequence is highly consistent with the original template. For example, when cloning some human disease – related genes, using the PFU enzyme for PCR amplification can avoid introducing unnecessary mutations and ensure the accuracy of the cloned gene, laying the foundation for subsequent gene function research and therapeutic applications.
2. Long – fragment DNA Amplification
   • For the amplification of long – fragment DNA (such as DNA fragments larger than 5 kb), the PFU enzyme shows unique advantages. Due to its good thermal stability and continuous synthesis ability, it can stably catalyze the DNA synthesis reaction at high temperatures, reducing the failure of amplification caused by the detachment of the polymerase. This makes the PFU enzyme an ideal choice in research such as the construction of large – fragment gene libraries and chromosome walking.

二、Accurate DNA Sequencing

1. Sanger Sequencing
   • In traditional Sanger sequencing reactions, the PFU enzyme can accurately synthesize complementary strands based on the template DNA. Its high – fidelity characteristic can reduce base mismatches, thereby improving the accuracy of sequencing results. In genome – sequencing projects, especially when sequencing complex genomes (such as plant and animal genomes), using the PFU enzyme can obtain more reliable sequence information, which is helpful for accurately analyzing gene structures and functions.
2. Next – generation Sequencing (NGS) Library Preparation
   • During the preparation of NGS libraries, it involves the amplification and processing of DNA fragments. The PFU enzyme can be used to ensure the accuracy of DNA synthesis in these steps. Accurate library preparation is crucial for subsequent high – throughput sequencing and data analysis. Using the PFU enzyme can reduce sequencing biases and misinterpretations caused by DNA synthesis errors.

三、Site – directed Gene Mutation

1. Introduction of Single – point Mutation
   • When studying the function of a specific position of a gene, it is often necessary to introduce a single – point mutation. The PFU enzyme can precisely introduce mutations at the target position and proofread the newly synthesized DNA strand by its 3′ – 5′ exonuclease activity to prevent additional random mutations. For example, when studying the relationship between protein structure and function, constructing single – point mutants with the PFU enzyme can accurately analyze the impact of a single amino acid change on the overall function of the protein.
2. Construction of Multiple – point Mutations
   • When it is necessary to construct mutations at multiple specific positions in a gene, the PFU enzyme is also applicable. It can accurately synthesize DNA strands with multiple mutation sites as designed, which is used for research such as constructing mutant gene libraries, helping to deeply understand the impact of the coordinated action of multiple sites in a gene on gene function.

四、Construction of Gene Expression Vectors

1. Studies on Promoters and Enhancers
   • When constructing gene expression vectors, research on regulatory elements such as promoters and enhancers often requires precise manipulation of DNA sequences. The PFU enzyme can be used to accurately clone and modify DNA fragments related to these regulatory elements to ensure the accuracy of their sequences. For example, when studying the impact of different promoters on gene expression intensity, using the PFU enzyme to construct expression vectors containing different promoters can accurately evaluate the functions of the promoters.
2. Optimization of Expression Vectors
   • In order to improve the expression efficiency of genes in host cells, it is necessary to optimize the expression vectors. The PFU enzyme can be used to make precise adjustments and modifications to gene sequences, transcription terminators, and other elements on the vectors. By accurately modifying these elements, the transcription and translation efficiencies of genes can be improved, thereby achieving efficient gene expression.