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Updated: Feb 16, 2026

Recombineering Homologous Recombination Constructs in Drosophila

Recombineering Homologous Recombination Constructs in Drosophila

Published on: July 13, 2013

Using overlap-extension PCR technique to fusing genes for constructing recombinant plasmids.

Yuanyuan Lu¹, Shuqin Xiao¹, Mingyue Yuan¹

¹College of Plant Protection, Shenyang Agricultural University, Shenyang, Liaoning, P.R. China.

Journal of Basic Microbiology

|January 3, 2018

Summary

This study simplifies overlap-extension PCR for gene splicing. The revised one-step method uses gradient temperatures and optimized ratios to efficiently create fused gene fragments for recombinant plasmids.

Keywords:

fragment concentration and ratio overlapping-PCR splice genes thermo-cycling condition vector construction

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Recombineering Homologous Recombination Constructs in Drosophila

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Use of In Vivo Assembly for High-efficiency Plasmid Construction

Use of In Vivo Assembly for High-efficiency Plasmid Construction

Published on: February 7, 2025

Area of Science:

Molecular Biology
Genetic Engineering

Background:

Overlap-extension PCR is a complex method for splicing gene segments.
This complexity limits its application in constructing recombinant plasmids.

Purpose of the Study:

To simplify the overlap-extension PCR protocol.
To improve the effectiveness of gene segment splicing for recombinant plasmid construction.

Main Methods:

Employed gradient temperatures instead of a single annealing temperature in thermocycling.
Optimized template concentration and fragment concentration ratio (inverse of fragment size ratio).
Adjusted fragment concentration to 10 ng/µl.

Main Results:

Successfully produced fused gene segments ranging from 2000-5000 bp.
Developed a revised one-step method for efficient gene splicing.
Demonstrated effective splicing of two or more fragments into a fused gene.

Conclusions:

The revised one-step method simplifies overlap-extension PCR.
This improved method enhances the efficiency of generating fused genes for recombinant plasmid construction.