Ligation of Sticky and Blunt End

Bacterial restriction enzymes are proteins that cut double-stranded DNA, cleaving phosphodiester bonds at specific sites, which are often referred to as restriction sites. These enzymes are also known as restriction endonuclease or molecular scissors. The term restriction comes from the fact that these enzymes restrict the entry of bacteriophage, which infects a bacterial cell by inserting its DNA into the bacterial cell for replication of the DNA.

Discovery

Restriction enzyme was first discovered in 1978 by Swiss microbiologist Werner Arber along with Stuart Linn. He discovered it while studying the phenomenon Host controlled restriction of bacteriophage. In 1970, Hamilton Smith and his co-worker first isolated a restriction enzyme from the bacterium Haemophilus influenzae strain.

Nature of Restriction Enzymes

The Restriction sites are mostly 4 to 6 bases long, and they are palindromic in nature, which means both forward and reverse strands have the same sequence. For example, Hind III is a Haemophilus influenzae restriction enzyme that recognises the sequence 5'AAGCTT-3' (upper strand) / 3'TTCGAA-5' (lower strand) and cleaves between the two A's on both strands.

Nomenclature

The name of any restriction enzyme consists of three parts.

1. An abbreviation of the genus and species of the organism to three letters, for example, Eco- for Escherichia coli identified by the first letter of the genus and the first two letters of the species.

2. A letter, number, or combination of the two to indicate the strain of the relevant strain.

3. A Roman numerical to indicate the order in which different restriction-modification systems were found in the same organism or strain
   
   

For example, the name EcoRI restriction enzyme was derived as

E – Escherichia (genus)

Co – coli (species)

R – RY13 (strain)

I – first identified (order of identification in the bacterium)

Sites of Cleavage

Rather than cutting DNA indiscriminately, a restriction enzyme cuts only double-helical segments that contain a particular nucleotide sequence, known as recognition sequence. The recognition sequence for the majority of restriction endonucleases is normally palindromes. Restriction enzymes make either blunt or staggered cuts. Thus, restriction fragments may have:

• Blunt ends are where the cleavage occurs at the middle of the target sequence and leave no overhangs.

• When the cleavage sites do not coincide with the symmetry axis, overhanging ends occur, resulting in so-called 5' or 3' overhangs on the restriction pieces. Overhanging ends produced by cleavage with a restriction nuclease are frequently referred to as sticky ends or cohesive termini.

DNA Ligase

Ligase can combine two sections of DNA that have identical ends to form an unbroken molecule. Ligase catalyses a reaction in which the hydroxyl group protruding off the 3' end of one DNA strand is connected to the phosphate group sticking off the 5' end of the other using ATP as its energy source. A sugar-phosphate backbone that is intact is created by this reaction.

Linkers and Adapters

Linkers are short sections of double-stranded DNA with a recognised nucleotide sequence that are 8–14 bp in length and have a location for 3–8 restriction enzymes to bind to them. By using ligase, these linkers are joined to blunt-end DNA. When compared to blunt-end ligation of large molecules, ligation is particularly effective because of the high concentration of these small molecules in the process. Cohesive ends are created by digesting DNA with the proper restriction enzyme, which cleaves the linkers' restriction sites to create the ends.

Adapters are linkers with cohesive ends. The idea of developing adaptors is to ligate the blunt of the adaptor to the blunt ends of the DNA fragment and to produce a new molecule with sticky ends.

Blunt End Ligation

Some restriction endonucleases have the ability to cut DNA at the opposing bases, resulting in DNA fragments with blunt ends. They are referred to as "blunt end cutters" because they don't leave single-stranded overhanging bases behind as they cleave down to the restriction site's centre. EcoRV HaeIII, AluI, and SmaI are often used as restriction enzymes for blunt end cutting. Two blunt ends are linked by a blunt end ligation. Compared to sticky end ligation, this ligation is less effective. For a blunt end ligation, the complementary ends of the DNA are not necessary.

Sticky End Ligation

dsDNA can be cut by some restriction endonucleases, leaving an overhanging fragment of single-stranded DNA at the end. Because sticky ends have unpaired bases and need complementary bases to establish bonds, sticky end ligation happens between two DNA fragments that have matching overhangs. In order to create identical ligating fragments from both DNA sources, it is necessary to use the same restriction enzyme. In cloning procedures, sticky end ligation is more effective and very desirable. EcoRI, BamHI, and HindIII are often used as restriction enzymes for sticky end cutting.

Difference Between Blunt and Sticky End Ligation

Interesting Facts

• The host-controlled modification pathway of pathogen resistance includes restriction enzymes.

• Type I and Type III restriction enzymes are bifunctional in nature with both methylase and endonuclease activity

• zinc finger nucleases falls under the category of artificial restriction enzyme

Key Features

• Restriction enzymes are proteins that cut DNA at specific sites leaving blunt or staggered ends.

• Sticky ends have unpaired bases at the end of the fragment, whereas blunt ends produce straight cleavage.

• Ligase enzyme can be used to bind two ends of the DNA fragments, which can be either blunt end or sticky end.

• EcoRV HaeIII, AluI, and SmaI are often used as restriction enzymes for blunt end cutting.

• EcoRI, BamHI, and HindIII are often used as restriction enzymes for sticky end cutting.