Difference Between Cloning and Subcloning

Key Difference – Cloning vs Subcloning
 

Cloning and Subcloning are molecular biological procedures which create genetically identical cells or organisms bearing the DNA or gene that are of interest. Cloning is a technique which involves insertion of the interested gene or DNA into a vector, replication of it within a host bacterium, and production of cells or organisms which are exact copies of the genetic makeup. Subcloning is a technique that involves insertion of the gene of interest, which is already inserted into a vector, into a secondary vector, replication of it within a host bacterium, and production of genetically identical copies of cells or organisms. The key difference between cloning and subcloning is that, in cloning, the gene of interest, once ligated into a vector, continue the cloning process while, in the subcloning, the already cloned gene of interest is separated from the parent vector and inserted again into a recipient vector and continue the process.

CONTENTS
1. Overview and Key Difference
2. What is Cloning
3. What is Subcloning
4. Side by Side Comparison – Cloning vs Subcloning
5. Summary

What is Cloning?

Cloning is the procedure which produces genetically identical organisms or cells. In nature, cloning happens in the means of asexual reproduction. When there is no genetic recombination or alteration, daughter cells receive same genetic makeup of the parent. Prokaryotic and eukaryotic organisms create clones by binary fission, budding, mitosis, etc.  In molecular biology, cloning genes or specific fragments of DNA is a popular method to study the structure and function of that particular DNA section.

The main objective of molecular cloning is to make millions of copies of genetically identical cells or organisms bearing the DNA fragment of interest (mainly genes). It creates organisms with exact genetic copies of another. Primarily, specific genes are cloned in molecular studies to obtain structural and functional information and for DNA sequencing. Also, for the production of specific proteins or products in large scale, cloning is widely used.

Cloning Procedure

The basic steps of cloning procedure are as follows.

1. Identification and isolation of the gene of interest. (Amplification of the gene of interest by PCR).
2. Restriction digestion of the Gene of interest (Restriction endonuclease cut the gene).
3. Restriction digestion of the vector DNA. (Vector DNA is also cut using the same restriction endonuclease).
4. Insertion of the gene into the vector and formation of the recombinant molecule.
5. Transformation of the recombinant vector into a host bacterium.
6. Isolation and identification of the transformed bacteria (plasmid vector should contain a selectable gene, most commonly an antibiotic resistance gene to screen transformed bacteria).
7. Recombinant gene expression within the host.

What is Subcloning?

Subcloning is a procedure of moving a gene of interest from one vector to another vector to see the expression of the gene to gain the desired functionality of the gene. In this method, two vectors are involved; namely, parent vector and destination vector. Cloned inserts are moved again into a second vector in subcloning. The objective of transferring the gene from the first vector to the second vector is to obtain something which could not be done by the first vector or to separate a gene again within the already cloned fragment of DNA and express it alone. Restriction enzymes are used in this procedure at the beginning.

Subcloning Procedure

The basic steps of subcloning are as follows.

1. With the help of restriction endonucleases, separation of the DNA of interest in the donor plasmid (parent vector).
2. Amplification of the DNA of interest using PCR.
3. Purification of the PCR product (DNA of interest) by gel electrophoresis.
4. Opening of the recipient plasmid by same restriction endonucleases used to separate DNA of interest in the parent plasmid.
5. Ligation of the DNA of interest (gene) into the recipient plasmid to create the subcloned plasmid.
6. Transformation of the subcloned vector into a competent host bacterium.
7. Screening of transformed cells.
8. Purification of the plasmid DNA and use for DNA sequencing or expression of the genes to obtain the desired products.

Subcloning is carried out on occasions of isolating one gene from the cloned group of genes or when the gene of interest is required to transfer into a useful plasmid to see the exact function of the gene of interest.

What is the difference between Cloning and Subcloning?

Summary – Cloning vs Subcloning

Cloning creates genetically identical cells or organisms with the inserted gene or DNA of interest. It proceeds through the separation and insertion of the DNA of interest into a vector and expression within a host bacterium. Subcloning shares similar steps with cloning. However, in subcloning, already cloned DNA fragment (gene of interest) is inserted into a vector and transformed into a host bacterium. That is the key difference between cloning and subcloning.

References

1. Lodish, Harvey. “DNA Cloning with Plasmid Vectors.” Molecular Cell Biology. 4th edition. U.S. National Library of Medicine, 01 Jan. 1970. Web. 05 Mar. 2017
2. “Subcloning.” Wikipedia. Wikimedia Foundation, 14 Feb. 2017. Web. 06 Mar. 2017
3. “Subcloning.” Subcloning | Open Access articles | Open Access journals | Conference Proceedings | Editors | Authors | Reviewers | scientific events. N.p., n.d. Web. 06 Mar. 2017
4. Wackerhage, Henning. “How to subclone.” How to subclone. N.p., 01 Jan. 1970. Web. 06 Mar. 2017

Image Courtesy

1. Molecular Cloning Procedure – By CNX OpenStax [CC BY 4.0], via Wikimedia Commons
2. Subcloning Procedure – By The original uploader was Takometer at English Wikipedia (Transferred from en.wikipedia to Commons.) [CC BY 2.5], via Wikimedia Commons