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Updated: Mar 23, 2026

Subcloning Plus Insertion SPI - A Novel Recombineering Method for the Rapid Construction of Gene Targeting Vectors

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Published on: January 8, 2015

Rapid and Robust PCR-Based All-Recombinant Cloning Methodology.

Abhishek Anil Dubey¹, Manika Indrajit Singh¹, Vikas Jain¹

¹Microbiology and Molecular Biology Laboratory, Department of Biological Sciences, Indian Institute of Science Education and Research (IISER), Bhopal, India.

|March 24, 2016

Summary

This study introduces a novel PCR-based cloning method that eliminates post-PCR modifications and screening. This rapid molecular cloning technique streamlines gene cloning and protein expression in E. coli.

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Area of Science:

Molecular Biology
Biotechnology
Synthetic Biology

Background:

Traditional cloning methods involve multiple post-PCR steps like restriction digestion and phosphorylation.
Screening for recombinant clones is often time-consuming and labor-intensive.
There is a need for faster and more efficient gene cloning and expression methodologies.

Purpose of the Study:

To develop a streamlined, PCR-based cloning methodology.
To eliminate the need for post-PCR modifications and clone screening.
To enable rapid gene cloning and subsequent protein expression.

Main Methods:

A two-step Polymerase Chain Reaction (PCR) approach is employed.
The first PCR amplifies the gene of interest.
The second PCR fuses the amplified gene with designed expression vector fragments carrying selection elements.

Main Results:

The method yields only recombinant clones, eliminating screening.
The entire process, including cloning and expression, can be completed in under 8 hours.
Successful cloning and expression of GFP, adh, and rho genes were demonstrated.
E. coli T7 express strain can be used directly for immediate protein expression post-PCR.

Conclusions:

This novel PCR-based cloning method offers a significant improvement in speed and efficiency.
The methodology simplifies the cloning process by removing the need for restriction digestion, phosphorylation, and screening.
The technique is versatile and applicable for direct protein expression, with or without affinity tags, in molecular and structural biology applications.