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:
- The DNA fragment of interest to be replicated
- 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.
The first step in determining how your ends will be blunted is to determine if they are 5´ or 3´ overhangs. This tutorial will teach you how to identify what type of overhang you have, as well as which enzyme will blunt that end, and how.
Transformation is the process by which bacteria are made to take up exogenous DNA. The word is derived from Griffith's discovery of a "transforming principle". Learn more about transformation and how it is used in cloning workflows.
Phosphorylation is the process by which phosphate groups are added to a molecule by a kinase. The phosphorylation status of a fragment of DNA can influence its ability to proceed in reactions. Learn more about phosphorylation and kinases.
Dephosphorylation is the process by which phosphate groups are removed from a molecule by a phosphatase. Removal of phosphate groups from a DNA fragment can prevent ligation. Learn more about dephosphorylation and phosphatases.
What are toxic mini-genes, and how do they improve transformation efficiencies? Becky explains.
For the inside scoop on how NEB products come to be, learn the story behind the new NEB® PCR Cloning Kit.
PCR Cloning is an easy and reliable cloning method. The name is derived from the use of a DNA amplification step to generate the amplicon. Learn more about the benefits and disadvantages of PCR Cloning.
Restriction enzymes are an integral part of the cloning workflow, for generating compatible ends on fragments and vectors. This animation discusses three guidelines for determining which restriction enzymes to use in your cloning experiment.
Ligation, the process of joining DNA fragments with a DNA ligase, proceeds in three steps. Learn more about ligation with our quick animation.
Traditional Cloning refers to the generation of DNA fragments using restriction enzymes, and their subsequent assembly and transformation. The name is derived from the method’s history as the first widely-accepted cloning method. Learn more about the benefits and disadvantages of Traditional Cloning.
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.
Learn more about the various types of molecular cloning found in the workflow below: Traditional Cloning, PCR Cloning, Seamless Cloning, Ligation Independent Cloning (LIC) and Recombinational Cloning.
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 and Gibson 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.
Learn more at NEBuilderHiFi.com.
FAQs for Cloning & Synthetic Biology
Protocols for Cloning & Synthetic Biology