Brief guide to gene cloning

Abstract

Analysis and manipulation of DNA is fundamental to understand gene function and expression. Gene cloning is a routine and versatile technique for molecular biology, allowing isolation, amplification, and production of recombinant DNA molecules. Here, we provide an overall process, various types, and applications of gene cloning. This concise guide will be useful for researchers who are unfamiliar with gene cloning, focusing on key principles and experimental considerations for efficient DNA analysis.

Keywords: Cloning; Ligation; Restriction enzyme; Selection; Transformation.

Fig. 1

Overview of gene cloning workflow. (A) Insert and vector preparation. Genomic DNA (gDNA) and complementary DNA (cDNA) that is reverse-transcribed from messenger RNA (mRNA) are two major types of inserts. The vector usually consists of the origin of replication (Ori), selectable marker, and multicloning site (MCS). (B) Recombination. The insert and the vector molecules are incorporated to generate recombinants. Ligation-dependent cloning methods include traditional cloning, Golden gate assembly, and TA cloning. Traditional cloning utilizes restriction enzymes and DNA ligase for recombination. In the Golden gate assembly, type IIS restriction enzyme digests the sequence distant from the recognition sites, followed by DNA ligase-mediated ligation. TA cloning recombines a T-tailed vector with an A-tailed insert. Ligation-independent cloning methods include Gibson assembly and Gateway cloning. In Gibson assembly, two or more DNA fragments with overlapping homologous ends are incorporated into a single vector. In Gateway cloning, site-specific recombination occurs between attachment (att) sequences during two steps: the BP reaction generates an entry clone by replacing the ccdB gene in the donor vector, and the LR reaction produces an expression clone by recombining the entry clone with a destination vector. (C) Transformation and selection. The recombinant DNA molecules are transformed into competent cells with heat shock or electroporation. Various selection methods are used to identify successfully transformed cells, including antibiotic resistance, blue-white screening, colony polymerase chain reaction (PCR), restriction mapping, and Sanger sequencing.