What is the Difference Between Single Digested Plasmid and Double Digested Plasmid

The main difference between single digested plasmid and double digested plasmid is that single restriction enzymes result in a single digested plasmid whereas two different types of restriction enzymes result in a double digested plasmid. Furthermore, the insertion of the foreign DNA fragment can occur in both orientations with single digested plasmids while the insertion of the foreign DNA fragment only occurs in the proper orientation with double-digested plasmids.

Single-digested plasmid and double-digested plasmid are two types of plasmids used in the recombinant DNA technology to produce recombinant DNA.

Key Areas Covered

1. What is a Single Digested Plasmid
     – Definition, Self-Ligation, Orientation
2. What is a Double Digested Plasmid
     – Definition, Double Digestion, Orientation
3. What are the Similarities Between Single Digested Plasmid and Double Digested Plasmid
     – Outline of Common Features
4. What is the Difference Between Single Digested Plasmid and Double Digested Plasmid
     – Comparison of Key Differences

Key Terms

Double Digested Plasmid, Double Digestion, Orientation, Recombinant Plasmid, Single Digested Plasmid, Single Digestion

What is a Single Digested Plasmid

Single-digested plasmids are a type of plasmids digested only with a single restriction enzyme. Hence, this process is called single digestion. Generally, plasmids used in recombinant DNA technology only contains a single restriction site for a particular restriction enzyme. Therefore, single digestion makes the circular plasmid linear, yielding a single DNA fragment. The two ends of this DNA fragment are compatible with each other; therefore, they can undergo self-ligation during the subsequent ligation with the insert. 

Figure 1: Single Digested Plasmid

Here, the insert also has to be digested with the same restriction enzyme to produce compatible ends with the plasmid. Therefore, the ligation occurs in two possible orientations: in the 5’ to 3’ orientation and 3’ to 5’ orientation. However, the inserts with the same orientation as the promoter region can undergo transcription. Thus, this orientation is known as proper orientation. Before proceeding, the recombinant plasmids with proper orientation has to be selected by screening the transformed colonies, either by PCR or biochemical assays.

What is a Double Digested Plasmid

Double-digested plasmids are the plasmids that undergo double digestion with two restriction enzymes. Here, this reaction can be carried out in a single tube. The restriction sites for both digestions occur within the restriction recognition site in the plasmid. Hence, double digestion reaction also results in a single DNA fragment. But, each of the two ends is produced by a different restriction enzyme. Therefore, these ends are not compatible with each other and do not undergo self-ligation.

Figure 2: Double Digested Plasmid

Most importantly, the insert can be ligated in the proper orientation with double-digested plasmids by digesting the ends of the insert to ligate in the proper orientation. Therefore, double-digested plasmids only yield the recombinant plasmids with proper orientation, which makes the subsequent selection of the orientation unnecessary. This saves time since it reduces a complete step from the recombinant DNA production procedure.

Similarities Between Single Digested Plasmid and Double Digested Plasmid

• Single-digested plasmid and double-digested plasmid are two types of plasmids used in recombinant DNA technology.
• Both single digestion and double digestion can be done by a single step reaction.
• Also, in both, the foreign DNA fragment is inserted between the two ends of the digested plasmids.
• Furthermore, both single and double digestion of a plasmid yield a single DNA fragment while two DNA fragments occur when a recombinant plasmid is digested.

Difference Between Single Digested Plasmid and Double Digested Plasmid

Definition

Single-digested plasmid refers to a plasmid digested by a single restriction enzyme while double-digested plasmid refers to a plasmid digested by two different restriction enzymes. These definitions explain the main difference between single digested plasmid and double digested plasmid.

Ends

In single-digested plasmids, digestion with the same restriction enzyme produce both ends while, in double-digested plasmids, digestion with a different restriction enzyme produce each end. Hence, this is an important main difference between single digested plasmid and double digested plasmid.

Self-Ligation

Furthermore, self-ligation is a major main difference between single digested plasmid and double digested plasmid. The ends of single-digested plasmids are compatible with each other for self-ligation while the ends of double-digested plasmids are not compatible with each other for self-ligation.

Importance

Moreover, insertion of foreign DNA fragment may occur in both orientations in the single-digested plasmids while the insertion of foreign DNA fragment can be done in the proper orientation in the double-digested plasmids.

Types of Recombinant Plasmids

A single-digested plasmid can produce three types of plasmids: two recombinant plasmids with opposite orientations and a self-ligated plasmid without an insert. Meanwhile, a double-digested plasmid can produce only one type of recombinant plasmid, which is in the proper orientation. Thus, this is another main difference between single digested plasmid and double digested plasmid. 

Time Saving

The recombinant fragments of single-digested plasmids have to be selected for their proper orientation while the double-digested plasmids ensure the proper orientation of the foreign DNA fragment. Therefore, double-digested plasmids save time in the recombinant DNA techniques, not single-digested plasmids.

Conclusion

Single-digested plasmids are digested with a single restriction enzyme, which results in compatible ends with self-ligation. Moreover, their ligation with the foreign DNA fragment can occur in both orientations. Therefore, recombinant plasmids with proper orientation have to be selected. On the other hand, double-digested plasmids are digested with two different restriction enzymes. Therefore, the two ends are not compatible for self-ligation. In addition, the foreign DNA fragment can be ligated in the proper orientation with double-digested plasmids. Therefore, the main difference between single digested plasmid and double digested plasmid is the type of digestion and the orientation of the insert.

Reference:

1. Griffiths AJF, Miller JH, Suzuki DT, et al. An Introduction to Genetic Analysis. 7th edition. New York: W. H. Freeman; 2000. Making recombinant DNA. Available Here