DNA Cloning
Overview
DNA cloning—often used interchangeably with molecular cloning—is an umbrella concept that includes many different experimental techniques whose goal is to isolate and expand a specific fragment of DNA into a host organism to create a large number of identical copies (recombinant DNA).
DNA cloning was first described in the 1970s and has since evolved to become the cornerstone of biotechnology.
What is DNA Cloning?
DNA cloning is comprised of multiple steps from generating recombinant DNA and vector constructs, to introducing them into host cells, to verifying the desired recombinant DNA final product. For this, DNA fragments are joined together and introduced into a vector which may include plasmids, attenuated viruses or bacteriophages, or larger constructs such as artificial chromosomes. Finally, the resulting recombinant DNA construct is transformed into host cells which use their own cellular machinery to replicate and express the construct.
This recombinant DNA technology is the foundation for a range of synthetic biology products like vaccines, biologics, enzymes, new crop varieties, microbial strains for bioremediation, research models, biodegradable materials, and biofuels, among others.
What is a DNA cloning workflow?
The basic cloning workflow involves these five steps:
- Designing and isolating the DNA sequence of interest (insert)
- Selecting the vector into which the insert will be introduced
- Using a cloning method to introduce the insert into the vector
- Transforming the cloned construct into host cells
- Screening and selecting host cell clones containing the correct recombinant DNA construct
Figure 1. An overview of cloning: A standard workflow for DNA assembly, transformation, and plating.
Types of cloning methods
There are numerous methods to insert a desired DNA fragment into a vector of choice. Each method has different applications and limitations—understanding which one is often a critical point in the experiment design step.
- Blunt-end cloning. Cloning of DNA fragments with blunt ends into a linearized vector using ligase to covalently link 3’-OH and 5’-PO4 ends.
- TA cloning. Cloning of DNA fragments into a vector with a Taq polymerase using TA-overhangs
- Restriction enzyme cloning. Cloning of DNA fragments into a vector with sticky ends produced by restriction endonucleases
- Golden Gate cloning. Cloning of DNA fragments into a vector using type IIS restriction enzymes that is based on complementing sticky ends
- Seamless cloning. Also known as Gibson Assembly®, seamless cloning of DNA fragments into a vector which is dependent on complementary overlaps at the terminal ends of the fragments and vector
- Gateway® cloning. Cloning technique that is recombination-based and allows shuffling DNA fragments between vectors
How are synthetic DNA fragments used for cloning and assembly?
Synthetic, double-stranded DNA fragments are convenient alternatives to PCR products and can be used in cloning and gene assembly applications. These materials are flexible and customizable, allowing users to design and validate in silico while also adapting the fragments to specific requirements of the cloning method. This means that these synthetic DNA sequences are a convenient and faster option because they do not require amplifying or assembling off various templates that must already be available in the lab.
IDT synthetic DNA fragment solutions for cloning methods
IDT offers three types of DNA fragments that are customizable and compatible with all cloning applications.
DNA Cloning Guide
This guide discusses common DNA cloning techniques, important sequence design considerations, and tips for troubleshooting that may arise when performing a cloning experiment.