Cloning & Synthetic Biology

Clone with Confidence^®

Whether you are performing your first cloning experiment or constructing multi-fragment DNA assemblies, NEB^® has the solution for you. Our high quality reagents are available for every workflow, including popular DNA assembly methods such as NEBuilder^® HiFi DNA Assembly and NEBridge^® Golden Gate Assembly. We also offer solutions for automation, site-directed mutagenesis, as well as your favorite restriction endonuclease, ligase, or competent cell products. When you are looking to clone with confidence, think of NEB. You can also use our free online tool, NEBcloner^®, to find the right products and protocols for each step in the cloning workflow.

Overview of molecular cloning

Molecular cloning refers to the process by which recombinant DNA molecules are produced and transformed into a host organism, where they are replicated. A molecular cloning reaction is typically comprised of the following two components:

1. The DNA fragment of interest to be replicated
2. A vector/plasmid backbone that contains all of the components for replication in the host

DNA of interest, such as a gene, regulatory element(s), or operon, etc., is prepared for cloning by excising it out of the source DNA using restriction enzymes, copying it using the Polymerase Chain Reaction (PCR), or assembling it from individual oligonucleotides. At the same time, a plasmid vector is prepared in linear form using restriction enzymes or PCR. The plasmid is a small, circular piece of DNA that is replicated within the host, and exists separately from the host’s chromosomal or genomic DNA. By physically joining the DNA of interest to the plasmid vector through phosphodiester bonds, the DNA of interest becomes part of the new recombinant plasmid and is replicated by the host.

Plasmid vectors allow the DNA of interest to be copied in large amounts and, often, provide the necessary control elements to be used to direct transcription and translation of the cloned DNA. As such, they have become the workhorse for many molecular methods, such as protein expression, gene expression studies, and functional analysis of biomolecules. During the cloning process, the ends of the DNA of interest and the vector have to be modified to make them compatible for joining through the action of a DNA ligase, recombinase, or in vivo DNA repair mechanism. These steps typically utilize enzymes, such as nucleases, phosphatases, kinases and/or ligases. Many cloning methodologies and, more recently, kits have been developed to simplify and standardize these processes, including those for seamless cloning and DNA Assembly.

In the article Molecular Cloning Technology – Past, Present and Future, read about the evolution of cloning technology—from early recombinant DNA experiments to advanced molecular cloning techniques—and learn how these innovations revolutionized genetics, biotechnology, and biomedical research.

Read the Feature Article

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History of molecular cloning

In 1952 with the genetic demonstration of phage restriction, the existence of mechanisms that protect bacteria from viral infections was revealed. This led to the isolation of the first restriction factor that could selectively cut bacteriophage DNA, in 1968. Over the following decades, scientists made significant advancements in understanding DNA replication and DNA modifying enzymes, leading to the development of various cloning techniques. Major milestones that revolutionized the field of molecular cloning during this time include the introduction of PCR in 1983, Golden Gate Assembly in 2008, and CRISPR-Cas9 gene editing in 2011.

This poster highlights a selection of some of the most significant discoveries, advancements, and achievements in the field of molecular cloning.

View PDF  Request a copy

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Types of molecular cloning

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Synthetic biology & DNA assembly

Synthetic Biology is a more recent expansion of the biotechnology field, in which genes and proteins are viewed as parts or devices, with the goal of re-designing and/or assembling these parts in novel ways to create a new and useful functionality. Recent advances in biofuels generation, production of biochemicals, and understanding the minimal genome all benefit from synthetic biological approaches. Often these projects rely on the ordered assembly of multiple DNA sequences to create large, artificial DNA structures. To this end, methods have evolved to simplify this process. NEBuilder HiFi DNA Assembly, Gibson Assembly^®, and NEBridge Golden Gate Assembly can be used to create many functional DNA structures, from a simple joining of two metabolic genes, all the way up to the creation of an artificial genome.

To help select the best DNA assembly method for your needs, please visit our Synthetic Biology/DNA Assembly Selection Chart.

Learn About DNA Assembly

Learn more about NEBuilder HiFi DNA Assembly at NEBuilderHiFi.com

Learn more about NEBridge Golden Gate Assembly at neb.com/GoldenGate

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