The importance of restriction enzymes lies in the property that it cleaves the DNA sequence, in most cases, within their specific recognition sequences unlike other restriction enzymes which cuts some base pairs away from their recognition sites. Some type II restriction endonucleases are also known to cleave the DNA sequence in close proximity of their recognition sequence rather than within the recognition site. This efficient nature of type II restriction endonucleases, combined with their comparatively smaller structure, has led to the wide application of these enzymes in gene cloning.
Mechanism of type II restriction endonucleases.
Recognition sites are specific area or sequences in the genetic molecule which these enzymes recognize as sites for cleavage. Recognition sites are unique for different restriction endonucleases. For type II restriction endonucleases, recognition sites are mostly palindromic sequences with rotational symmetry. DNA has a double stranded helical structure where, the nucleotides of the two strands of DNA are complementary to each other. There are certain sequences in such a structure where, the first half of the sequence is a mirror image of the second half of the complementary strand and reads identical from same end. Such sequences are termed as palindromic sequence with rotational symmetry.
The restriction endonuclease moves along the surface of the DNA until it recognises its target sites. After recognition, it initiates DNA binding in the presence of Mg2+ ions resulting in cleavage at specific sites.
The cleavage patterns produced by different restriction endonucleases are specific and each holds a novel role in gene cloning.
The two main patterns of cleavage are creating staggered cuts and even cuts. In staggered cuts, the cleavage occurs in different locations resulting in producing protruding ends of one of the strands in the double helix. Such ends are known as cohesive or sticky ends. The main benefit of such ends is that the protruding ends created are usually complementary in nature and can be used to link with vectors consisting complementary sequences for isolating the DNA fragment. It forms the basics for recombinant DNA techniques such as southern blotting. The even cuts, on the other hand, produces blunt ends where the two strands are cleaved at similar points. The importance of blunt ends in gene cloning involves many techniques which are utilized to modify the blunt ends in a manner so as to meet the specific requirements.
Tailing: This is a procedure which results in a protruding end of a defined length being created which aids in the pairing of required DNA segment with appropriate vector.
Linker: Linkers are chemically synthesized oligonucleotides. This can be used to modify the blunt ends so as to create cohesive ends of required bases. Linkers are so designed as to have a recognition site of a specific endonuclease. This can be linked to a blunt end DNA fragment created by the restriction digest. Such a modified fragment when digested by the linker specific restriction endonuclease can create cohesive ends complementary to vectors which can later be isolated to create multiple copies or, can be used in creating a recombinant DNA.
Adapters: These are short artificially synthesized double stranded fragments which can be used to link two blunt ends with different end sequence.
As a result of all these techniques, it is possible to alter a specific gene at a nucleotide level by identifying the respective restriction endonuclease enzyme which can cleave at the specific site. This is the principle adapted in gene cloning given that, to create a specific clone it is required to isolate the target gene. This isolation can only be done with the help of a restriction endonuclease enzyme. The type II restriction enzymes accentuates the importance as they have the ability to cleave at exact points resulting in producing definite fragments rather than random fragments.
(i) RFLP (restricted fragment length polymorphism), which involves production of DNA fragments of different lengths which can be separated and utilized for several purposes like DNA fingerprinting, identification of mutations, preparation of genomic library etc.
(ii) A technique called restriction mapping make use of the capability of restriction enzymes to create DNA fragments of specific length thus distinguish alleles of a single gene having altered restriction sites.
(iii) Gene therapy: This employs the property of restriction endonuclease to recognize and remove a specific DNA fragment responsible for many diseases.