Key Difference – Blunt vs Sticky End Ligation
Restriction endonucleases are specific enzymes which cut double-stranded DNA (dsDNA). They are also known as molecular scissors in Molecular biology. Restriction enzymes are able to recognize specific short sequences of the dsDNA known as recognition sites and cleave phosphodiester and hydrogen bonds to open double strands. As a result of cleavage by these enzymes, DNA fragments are produced with different types of ends such as sticky ends and blunt ends. DNA ligase is an enzyme used in Molecular biology to join two adjacent strands of DNA by forming new bonds. The step is known as ligation and according to the type of the DNA end ligated, they can be referred to as blunt end ligation and sticky end ligation. The key difference between blunt and sticky end ligation is that blunt end ligation occurs between DNA fragments which contain two blunt ends whereas sticky end ligation occurs between 5’ and 3’ overhangs. Compared to blunt end ligation, sticky end ligation is more efficient and stable.
CONTENTS
1. Overview and Key Difference
2. What is Blunt End Ligation
3. What is Sticky End Ligation
4. Side by Side Comparison – Blunt vs Sticky End Ligation
5. Summary
What is a Blunt End Ligation?
Some restriction endonucleases can cut the DNA at the opposite bases and produce blunt end DNA fragments. These enzymes are known as blunt end cutters; they cleave straight down to the middle of the restriction site without leaving single stranded overhanging bases. Blunt ends are also known as non-overhanging ends since they do not have 3’ and 5’ overhanging bases at the ends. Both strands terminate from base pairs in blunt ends. Common blunt end cutting enzymes are EcoRV HaeIII, AluI and SmaI.
Blunt end ligation is involved between two blunt ends. It is not a ligation of protruding bases. This ligation is less efficient than sticky end ligation. However, in some occasions, blunt end ligation becomes more advantageous than sticky end ligation, especially when ligating PCR products. PCR products are always produced with blunt ends. Blunt end ligation does not require complementary ends in the DNA for the ligation.
Figure 01: Blunt end production by Eco RV enzyme
What is a Sticky End Ligation?
Some restriction endonucleases are able to cut dsDNA, leaving an overhanging piece of single-stranded DNA at the end. These ends are known as sticky or overhanging ends. Sticky end ligation occurs between two DNA fragments which contain matching overhangs because sticky ends are possessed with unpaired bases and require complementary bases to form bonds. Hence, it is necessary to use the same restriction enzyme for both sources of DNA to produce the matching ligating fragments.
Sticky end ligation is more efficient and is often highly desirable in cloning processes. There are several restriction enzymes which produce sticky ends. They are EcoRI, BamHI, HindIII etc.
Figure 02: Sticky end production by Eco RI enzyme
What is the difference between Blunt and Sticky End Ligation?
Blunt vs Sticky End Ligation |
|
| Blunt end ligation occurs between two blunt end DNA fragments. | Sticky end ligation occurs between two matching DNA fragments with sticky ends. |
| Enzymes | |
| Blunt end cutters produce blunt ends. | Sticky end cutters produce sticky or cohesive ends. |
| Requirement of Matching Ends | |
| It does not require matching fragments or complementary bases. | It requires complementary bases at the ends to form base pairs. |
| Efficiency | |
| It is less efficient than sticky end ligation | It is more efficient than blunt end ligation. |
Summary – Blunt vs Sticky End Ligation
Restriction endonucleases are able to cleave dsDNA and produce DNA fragments with different ends. They recognize specific sequences and restrict DNA creating sticky and blunt ends. Sticky ends have unpaired bases at the end of the fragments. Blunt ends are created due to a straight cleavage and they have base pairs at the ends. Sticky end ligation requires two complementary single-stranded DNA pieces. Blunt end ligation occurs between any two blunt end fragments. This is the difference between blunt and sticky ligation.
References:
1. Roberts, Richard J. “How restriction enzymes became the workhorses of molecular biology.” Proceedings of the National Academy of Sciences. National Acad Sciences, n.d. Web. 12 Apr. 2017
2. Adnan, Amna. Uses of Restriction Enzymes in Biotechnology. N.p., n.d. Web. 12 Apr. 2017
Image Courtesy:
1. “EcoRV Restriction Site.rsh” By Ramin Herati – Created with Inkscape (Public Domain) via Commons Wikimedia
2. “Restriction enzyme Eco RI” By Tinastella – Own work (Public Domain) via Commons Wikimedia