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.

INTRODUCTION

The ability to analyze and manipulate DNA is crucial for understanding the structure, function, and expression of genes. Advances in molecular biology techniques, including gene cloning, polymerase chain reaction (PCR), sequencing, and genome editing, have substantially contributed to the study of individual gene regulation, as well as various applications in medicine and industry (Abdellaoui et al., 2023, Bae et al., 2023, Heo et al., 2023, Jeong et al., 2023, Lee et al., 2024).

Among the DNA manipulation techniques, gene cloning remains an essential tool because of its versatility, reliability, and cost-effectiveness (Han et al., 2023, Kim et al., 2024a, Kim et al., 2024b, Kim et al., 2025, Lee et al., 2023, Nguyen et al., 2023, Sharma et al., 2014). Gene cloning is a technique that assembles the gene of interest into recombinant DNA molecules and replicates them in a host organism (Lodge et al., 2007). Here, we provide an overview of gene cloning, focusing on fundamental cloning steps, commonly used methods, and applications in molecular biology. By summarizing key principles and experimental considerations, this guide will serve as a valuable resource for scientists who employ gene cloning in their research.

MAIN BODY

Overview

The detailed protocol for gene cloning varies depending on the starting material. Genomic DNA (gDNA) can be isolated from cells or tissues by employing chemical, enzymatic, or mechanical lysis (Patil et al., 2022), whereas complementary DNA (cDNA) is reverse-transcribed DNA from messenger RNA (mRNA) (Harbers, 2008). Polymerase chain reaction (PCR) is used to amplify the insert DNA. Melting temperatures, the percentage of guanine (G) and cytosine (C) bases, the lengths of oligonucleotides, and secondary structures are the core parameters that ensure successful PCR primer design (Chen et al., 2002). Additionally, codon optimization is also an important element for improving the expression levels of recombinant DNA molecules because of variations in codon usage among different species (Menzella, 2011).

DNA vector is a type of storage system that can replicate autonomously within host cells, usually bacteria, such as Escherichia coli. Generally, the DNA vector has three characteristics: origin of replication (Ori), selectable marker, and multicloning site (Carter et al., 2022, Clark and Pazdernik, 2012) (Fig. 1A). In addition, the stability and efficiency of gene delivery depend on the insert size. Once the recombinant DNA is transferred to the host cells, the copy number and promoter strength of the vector determine replicon amplification (Preston, 2003).

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.

The obtained insert and vector DNA molecules are incorporated to generate recombinants. The recombination step can be classified into traditional ligation-dependent and ligation-independent processes (Ashwini et al., 2016, Yao et al., 2016). Here, we provide three types of ligation-dependent cloning methods and two types of ligation-independent cloning methods. Proper methods can be utilized depending on aims and characteristics of inserts and vectors (Fig. 1B, Table 1).

Table 1.

Features of various types of gene cloning

	Ligation-dependent cloning			Ligation-independent cloning
	Traditional cloning	Golden gate assembly	TA cloning	Gibson assembly	Gateway cloning
Restriction enzyme required	Yes	Yes (type IIS)	No	No	No
Insert preparation	Restriction enzyme digestion		PCR to generate A-tailed insert	PCR to generate overlapping homologous ends	PCR to generate att-flanked sequence
Vector preparation	Restriction enzyme digestion		Linearization to generate T-tailed vector	Linearization to generate overlapping homologous ends	Using donor vector and destination vector
Recombination	Ligation between 5′ and 3′ ends using DNA ligase			Assembly between overlapping homologous sequences	Site-specific recombination between att sites

PCR, polymerase chain reaction; att sites, attachment sites

After the inserts are incorporated into the vectors, the recombinant DNA molecules are introduced to competent bacterial cells via transformation (Johnston et al., 2014). Bacillus subtilis, Streptococcus pneumonia, Neisseria gonorrhoeae, and Haemophilus influenzae exhibit natural competence (Das and Dash, 2015). For other bacterial strains, including E. coli, additional work is required to generate competent cells. Heat shock and electroporation are two representative methods (Fig. 1C). The heat shock method is relatively economical and easy to perform in laboratories. Electroporation is approximately 10 times more effective than heat shock methods. However, specialized equipment, such as electroporators and cuvettes, is required (Chang et al., 2017, Das and Dash, 2015, Green and Sambrook, 2021, Kotnik et al., 2015, Warren, 2011). Researchers need to choose proper methods depending on aims and expected results.

Transformed bacterial cells are usually cultured on semisolid agar plates (Bertero et al., 2017). Screening whether the colonies contain the recombinant DNA and the orientation of the insert is correct is important for successful gene cloning. Antibiotic resistance is the simplest way to identify the presence of recombinant DNA molecules because, as mentioned above, one of the most important characteristics of vectors is the existence of a selectable marker. Blue-white screening is a method that utilizes the expression of the lacZ gene in E. coli (Das and Dash, 2015, Lodge et al., 2007). Colony PCR, restriction mapping, and Sanger sequencing are more direct and accurate methods to select transformants. For detailed principles and protocols, researchers can consult other resources (Andreou, 2013, Bertero et al., 2017, Jacobus and Gross, 2015, Goranson and Erbe, 2003) (Fig. 1C).

Cloning

Ligation-dependent cloning

For traditional ligation-dependent cloning, restriction enzymes recognize unique sites that have palindromic sequences, called recognition sites (Pelley, 2011). Researchers need to consider several criteria to choose the most appropriate restriction enzymes: fragment size, resulting ends of the cleavage, and methylation sensitivity (Brown, 2020, Carter et al., 2022, Robinson et al., 2004). After restriction enzyme digestion, vector and the insert DNA fragments are joined together (Fig. 1B). DNA ligase catalyzes the reformation of covalent phosphodiester bond between 5′-phosphyl group on one end and 3′-hydroxyl group at the other end. T4 DNA ligase and E. coli DNA ligase are the two most common reagents (Shi et al., 2018, Shuman, 2009, Wilkinson et al., 2005).

Golden gate assembly is a one step, one-pot cloning method that is based on the type IIS restriction enzymes (Engler et al., 2008, Engler and Marillonnet, 2014, Hsu and Smanski, 2018). The type IIS restriction enzymes, such as BsaI, BsmBI, and BbsI, cleave the sequence distance from the recognition sites. In addition, the original restriction sites are not present after the ligation, allowing seamless cloning (Ashwini et al., 2016, Bird et al., 2022, Pingoud et al., 2005). Golden gate assembly is simpler than traditional restriction enzyme digestion and ligation cloning in that it enables the incorporation of multiple fragments simultaneously (Kirchmaier et al., 2013, Weber et al., 2011). Moreover, the likelihood of further digestion by restriction enzyme and self-ligation of the vector is lower because the recognition sites are removed after the cleavage, and the ends are incompatible with each other (Luo et al., 2018).

TA cloning is one of the simplest and most commonly used form of PCR cloning methods (Zhou and Gomez-Sanchez, 2000). The terminal transferase activity of DNA polymerase adds a single deoxyadenosine (dA) residue at the 3' ends of the insert DNA fragments. The PCR-amplified products are then ligated with linearized T-vector, which has single-stranded T overhangs at the 3′ ends (Bertero et al., 2017, Clark and Pazdernik, 2012, Yao et al., 2016). In particular, TA cloning is useful when compatible recognition sites are not available in the insert and vector DNA molecules. In addition, minor modification, such as hemi-phosphorylation of both A-tailed inserts and T-tailed vectors, ensures unidirectional cloning (Maheshwari et al., 2022).

Ligation-independent cloning

Gibson assembly is an isothermal, single-reaction method to assemble multiple overlapping DNA fragments (Gibson et al., 2009). This process is achieved by adding homologous sequence at each end of the DNA fragments that are to be cloned (Avilan, 2023, Bertero et al., 2017, Wang et al., 2015). (Fig. 1B). The reagents for Gibson assembly are available from New England Biolabs (Gibson, 2011). The researchers can utilize other commercial cloning kits based on a similar principle, such as In-Fusion HD Cloning, GeneArt Seamless Cloning and Assembly, and Cold Fusion Cloning (Celie et al., 2016). Gibson assembly facilitates simple and efficient gene cloning (Chen et al., 2025). Moreover, additional modification enables researchers to assemble large DNA fragments with high GC contents (Li et al., 2018).

Gateway cloning is based on site-specific recombination used by bacteriophage lambda to integrate its DNA into the E. coli genome. Two sets of reversible reactions are performed in this method (Magnani et al., 2006). For the BP reaction, the insert fragments are incorporated into a donor vector to generate an entry clone. Next, the LR reaction is a process of constructing an expression clone by combining the entry clone with the destination vector (Ashwini et al., 2016, Clark and Pazdernik, 2012, Hartley et al., 2000). The BP and LR reactions are mediated by recombination between specific attachment (att) sites, during which the toxic ccdB gene in the donor or destination vector is replaced by the insert DNA, allowing only correctly recombined clones to survive (Bachman, 2013, Reece-Hoyes and Walhout, 2018, Walhout et al., 2000). Once an entry clone is generated, Gateway cloning allows large-scale cloning into various types of destination vectors in a standardized manner (Luo et al., 2018). In addition, multisite Gateway cloning enables researchers to assemble multiple insert DNA fragments into a single construct in a definite order and orientation (Sasaki et al., 2004).

Applications

Site-directed mutagenesis

Site-directed mutagenesis is a molecular biological technique to introduce specific nucleotide changes into the gene of interest, allowing the study of structure-function relationships, genome modifications, and/or protein interactions (Bachman, 2013, Carrigan et al., 2011, Goutam et al., 2024, Kim et al., 2023, Um et al., 2023, Zhang et al., 2021). Among various methods, PCR-based site-directed mutagenesis is widely used to yield mutations, including substitutions, insertions, or deletions. (Lee et al., 2010, Lodge et al., 2007, Wong, 2006). Advances in PCR-based mutagenesis, such as overlap extension PCR, inverse PCR, and megaprimer PCR, exhibit high efficiency and facilitate mutagenesis of large DNA fragments (Zawaira et al., 2012).

DNA library

DNA library is a collection of randomly fragmented DNA molecules that are cloned and/or screened for typical phenotypes (Patil et al., 2022). The genomic library consists of DNA fragments that represent an organism’s entire genome, and is mainly used to analyze genetic mutation as well as for genome sequencing or genome mapping (Clark and Pazdernik, 2012, Singh et al., 2021). For the cDNA library, total RNAs extracted from biological samples are reverse-transcribed using oligo(dT) primers (Harbers, 2008, Nam et al., 2002). The cDNA library is used to identify gene expression patterns in a typical cell type or tissue. Moreover, a labeled cDNA library can also be utilized to compare transcriptomic changes in various diseases (Brown, 2020, Imdad et al., 2024).

CONCLUDING REMARKS

This MiniResource provides a brief overview of gene cloning for DNA analysis and manipulation, from the overall process and different methods for recombination to applications. Each cloning strategy has its respective strengths and limitations. Traditional ligation-dependent cloning methods rely on restriction enzyme digestion and ligation, allowing cost-effective cloning, but are restricted by the availability of restriction sites. Ligation-independent cloning methods offer advanced, seamless, and high-throughput assembly, particularly for assembling multiple fragments, but are often limited by time-consuming primer design or the use of costly reagents (Ashwini et al., 2016, Lodge et al., 2007). Researchers need to choose and modify proper methods depending on the characteristics of DNA molecules and aims. For more comprehensive guidelines, users are encouraged to refer to additional publications, particularly detailed protocols of various gene cloning methods (Brown, 2020, Engler and Marillonnet, 2014, Gibson, 2011, Green and Sambrook, 2012, Maheshwari et al., 2022, Reece-Hoyes and Walhout, 2018).

FUNDING AND SUPPORT

This work was supported by the National Research Foundation of Korea grant funded by the Korea government (MSIT) (RS-2024-00408712) to S.J.V.L.

AUTHOR CONTRIBUTIONS

W. H., S.G.H., H.C.K., and S-J.V.L. wrote the manuscript.

CRediT authorship contribution statement

Woojin Hong: Writing – review & editing, Writing – original draft, Conceptualization. Seung-Jae V. Lee: Writing – review & editing, Writing – original draft, Conceptualization. Seokjun G. Ha: Writing – review & editing, Writing – original draft, Conceptualization. Hyunwoo C. Kwon: Writing – review & editing, Conceptualization.

Declaration of generative AI and AI-assisted technologies in the writing process

During the preparation of this work the authors used ChatGPT 4o (OpenAI) in order to improve language clarity of the manuscript. After using this tool, the authors reviewed and edited the content as needed and take full responsibility for the content of the publication.

DECLARATION OF COMPETING INTERESTS

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.