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Role of Type 2 Restriction Endonucleases in Gene Cloning
Gene cloning involves application of many enzymes having specific functions to result in a modified product. Of the different enzymes involved, endonucleases are a type of enzymes which have the ability to cut or cleave the DNA molecule. Restriction endonuclease refers to a group of endonucleases which cleaves the DNA at specific points known as recognition sequences or sites. Different types of restriction endonucleases have been identified like type I, II, and III. Among these, the most available and most extensively used enzyme is type II restriction endonuclease. Examples are: ECoR I, Hind III, etc.

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.

These include:
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.

Other Applications:
(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.
Very well written. Simplified and well composed with all necessary details.
Alkaline phosphatase: The main function is dephosphorylation. This enzyme is used to remove the phosphate group from the 5-prime end of DNA and RNA. The DNA becomes linear on dephosphorylation.

Poly nucleotide Kinase: Catalyzes phosphorylation thus adds phosphate group to double stranded or single stranded nucleic acids at its 5-prime OH end with the help of ATP.

DNA Ligase: Ligates the DNA strands with the help of phospho diester bond. Single strand breaks are also repaired by this enzyme.

Restriction Enzymes: This enzyme is restriction site specific and cleaves both the strands of a double stranded DNA. It helps in the formation of the 5-prime phosphate end and 3-prime hydroxyl end of a DNA. EcoR I, EcoR II, BamH I, Hind III are some of the examples of restriction enzymes.

DNA polymerase I: Involved in DNA replication. Used in the generation of complementary DNA in 5-prime to 3-prime direction from a DNA template.

Taq DNA polymerase: This enzyme obtained from Thermus aquaticus bacterium is used in DNA amplification (PCR).

RNA polymerase: The different RNA polymerases are T7, T3 and SP6 obtained from the respective bacteriophages and they recognize only phage specific promoters in the process of DNA transcription.

RNase A: RNA specific enzyme and cleaves only RNA

RNase H: Acts only upon RNA in a hetero duplex structure comprising RNA-DNA thus completes the process of DNA replication.

Exonuclease III: Cleaves the DNA (linear) from its end. It acts upon double stranded DNA by always removing nucleotides from the 3-prime OH end only.

Mung bean nuclease (Endonuclease): This enzyme is obtained from Mung bean and hence the name mung bean endonucleases and it is single stranded DNA or RNA specific and acts on the same.

Nuclease S1 (Endonuclease): This enzyme is again strand specific and only digests single stranded DNA or RNA. It is used in the generation of blunt end DNA molecules by removing its single strand while synthesizing double stranded c DNA.

Reverse Transcriptase: Its functions include synthesis of cDNA using RNA template and this property of reverse transcriptase is used to create cDNA libraries.

Terminal Transferase: The role of this enzyme is addition of nucleotides to the 3-prime end of either single or double stranded nucleic acids. This enzyme is used in PCR technique and in rapid amplification of cDNA ends.

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Role of Type 2 Restriction Endonucleases in Gene Cloning31