Questions and Answers about Molecular Cloning

"Everyone is a writer" program aims to promote the sharing of knowledge and learning together. We encourage scientists, researchers, and students to answer the questions with their expertise and help others learn from the answers. There are some answers about the molecular cloning from this campaign.

Q1: What is molecular cloning?

A: Pratistha Satpathy: Molecular cloning is the collection of experimental procedures required to isolate and expand a specific fragment of DNA into a host organism in order to create a large number of identical copies. Molecular cloning is a powerful technique that permits the generation of complex combinations of DNA fragments. Learn More

Kevin Arcos: Molecular cloning refers to the replication and recombination of DNA molecules, which makes many identical copies of a piece of DNA, such as a gene of a genome, enabling the manipulation and study of genes. The workflow of cloning has four steps: Learn More

Emiliano Vilardo: Gene cloning for me is any technique that multiplies a gene. The ingredients are always a fragment of DNA with some gene and a circular DNA molecule called vector. First open the vector with restriction enzymes or in a pcr reaction with a very powerful polymerase that makes no mistakes, then mix both of them in a tube and you get your recombinant DNA molecule. Learn More

Q2: What are the necessary components of a plasmid?

A: Dean Niu: The plasmid used for cloning and expression usually including three major components: 1. Replicon: Also known as the replication initiation region, it contains genetic factors that control the origin of plasmid DNA replication and plasmid copy number. Learn More

Kevin Arcos: A plasmid contains three necessary functional regions: an origin of replication, a polylinker to clone the gene of interest (called multiple cloning site where the restriction enzymes cleave), and an antibiotic resistance gene (selectable marker).  Learn More

Pratistha Satpathy: 1. Origin of replication(ori): a required sequence or element on the plasmid for its replication inside the host. Also called as the replicon. 2. Selectable Marker: a required element for cloning to select a host, which carries the DNA construct. Learn More

Emiliano Vilardo: What a plasmid cannot lack is a replication origin to replicate inside the host cell and a selectable marker to tell which cells have engulfed it. Also a polylinker which is a cluster of cloning sites artificially made that allow restriction enzymes to cut and open the plasmid in those locations to receive the insert. Nowadays you can open your plasmid in any location if using Gibson assembly. Learn More

Q3: What's the difference between a plasmid and a vector?

A: Raghuvandhanan K S: PLASMIDS are extrachromosomal DNA molecules that can replicate independently. They are mostly circular and double-stranded. They carry beneficial genes responsible for  Antibiotic resistance, degrading Aromatic compounds etc.. eg: Ti Plasmids(Tumour inducing). VECTORS, These are vehicles, essentially used to deliver Nucleic acids(mostly DNA) to the desired host. Learn More

Pratistha Satpathy: A vector is a DNA molecule which can carry a DNA insert to generate a recombinant DNA and replicate in a particular host.

It is artificially engineered in labs. Also called constructs. Example: Cosmid, Artificial Chromosomes, Plasmid. Apart from containing ori, selectable marker, engineered plasmids have an additional cloning site. Learn More

Kevin Arcos: A plasmid is a small circular DNA element, which is capable to carry several genes. Plasmid size can be varied from less than 1.0 kb to more than 200 kb. A vector refers to a self-replicating DNA fragment, which can be used to link with another DNA fragment, creating recombinant DNA.  Learn More

Emiliano Vilardo: A vector is not always a plasmid. It can also be something else like the chromosome of a virus or any molecule that can carry a gene into a host cell which is the requisite to be a vector. A plasmid is just the most popular vector. Learn More

Q4: What are the facts that determine plasmid copy number?

A: Pratistha Satpathy: Plasmids are extrachromosomal DNA elements with characteristics copy number within the host. Although plasmid copy number may vary in different bacteria, within a given host, and under fixed growth conditions, any particular plasmid has a characteristics copy number. Learn More

Emiliano Vilardo: The time will come when you encounter protocols that ask you if your plasmid is low copy number or high. What is it and why is it important? The reasons for being a multicopy plasmid (those with guarantee that you will have at least 20 copies per cell) are still a mystery but a hint that you can be working with a low copy number plasmid is that yours is one of the larger ones. Learn More

Q5: How many DNA cloning methods have been developed?

A: Emiliano Vilardo: There is traditional restriction enzyme cloning and then there are the new methods which possess one big advantage: they are significantly cheaper than traditional cloning, in terms of both materials and time. You could say that there are many DNA cloning methods as ideas. Gibson Assembly from NEB is one of them, In-Fusion by Takara-Bio (formerly Clontech) is another one. Learn More

Q6: What vector can be used to clone a very large DNA fragment?

A: Pratistha Satpathy: The standard plasmid and phage lambda vectors can accept donor DNA of sizes as large as 25 to 30 kb. However, many experiments require inserts well in excess of this upper limit. To meet these needs, the following special vectors have been engineered. In each case, after the DNAs have been delivered into the bacterium ,they replicate as large plasmids. Learn More

Emiliano Vilardo: Plasmids are not sufficient to clone large fragments that is why if you would want to clone a big human gene of 100kb you will need to search other category of vectors which are based on yeast or bacterial chromosomes, yeast chromosomes called YACs can hold up 600kb easily. Learn More

Q7: What are the commonly used vectors for gene expression in prokaryotic and eukaryotic systems?

A: Emiliano Vilardo: Almost all nowadays plasmids have something left of one of the most famous plasmids: the pBR322. ‘p’ indicates that this is indeed a plasmid. ‘BR’ identifies the laboratory in which the vector was originally constructed. For protein expression in E. coli it is common to use the pBAD, pET, pHAT series which are derived from the beloved pBR322. For Eukaryotic systems like yeast the pYES2 vector is very common. Learn More

Q8: What are the free tools for plasmid design and plasmid mapping?

Emiliano Vilardo: The best way is to take a piece of paper and draw your plasmid map, then store it in a black carpet with the label “top secret” and stick it in a dusty bookshelf in between your labs collection of old Sigma-Aldrich catalogs. If you don't have any of that in your lab you can use online tools like Benchling or the online tool in the GenScript webpage. Learn More

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