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let's begin with why this enzyme is called as restriction enzyme. This is a bacterium, On the top of bacterium, there is a bacteriophage. Bacteriophage are viruses that attacks bacterium. upon attachment this bacteriophage injects its genetic material into the bacterium. bacteria has a defense mechanism in the form of an enzyme which is called as restriction enzyme that cut this viral genome making it inactive. As this enzyme restricts the replication of viral genome inside bacterium this enzyme is called as restriction enzyme. Bacteria's own DNA is protected from this enzyme by means of specific methylation. Restriction enzymes are enzymes that is capable of making cuts in a double-stranded DNA molecule at unique sequences called recognition sequences. so this is a restriction enzyme Hind III as you see, this cuts within this sequence as this enzyme is capable of cutting DNA molecule this enzyme is also called as molecular scissors, molecular knives and molecular scalpels. The discovery of restriction enzyme actually marked the beginning of recombinant DNA technology. Hind II is the first restriction endonuclease isolated from Haemophilus influenzae bacteria, here second indicates the second strain identified of this bacterium Haemophilus influenzae. Types of restriction enzymes. Restriction enzymes belongs to the class nucleuses or nucleic acid degrading enzymes there are two broad classes endonucleases and exonucleases as the term indicates n2 means these endonucleases makes cuts within the DNA molecule like this whereas exonucleases causes cuts at the ends or terminal regions or removing nucleotides from the ends endonucleases are widely used in recombinant DNA technology endonucleases there are three types of restriction and nucleases type 1 type 2 and type 3. Type 2 is widely used in recombinant DNA technology as it makes specific cuts within DNA molecule called as recognition site or restriction site. Examples include Eco R1, HindIII etc. Magnesium ions are required for cleavage. type 1 and type 3 makes random cuts so rarely used in recombinant DNA technology. How restriction enzymes cut DNA molecule so let's take Eco R1 as an example Eco R1 is a restriction enzyme isolated from Escherichia coli R1 strain, so this endonuclease recognizes a sequence a specific sequence GAATTC which is called as recognition sequence or restriction site. So, this is how this enzyme makes a cut. So, this is a recognition sequence whenever the sequence is there, this enzyme can make cuts within the sequence. This is the recognition sequence GAATTC, this will make cut after G and here it is before G before guanine nucleotide this side is called as recognition side these are the characteristics of recognition side it is often four to eight base pairs there are palindromic sequences as you see g a a t t c from five prime to three prime direction from here five prime to 3 prime direction it reads the same gaattc that's why they are palindromic sequences often with 4 to 8 base pairs they are called as recognition sites or restriction sites. then cut patterns by restriction enzymes. First one is sticky ends or staggered cuts, enzyme cuts that generate protruding single stranded ends or extensions as in the case of Eco R1. Let's take an example the enzymes that make sticky ends are preferred in recombinant technology. As you see this is Eco-R1 restriction site gaattc, so it will make cut like this so after the cut there are single stranded overhangs or single stranded extensions this type of cut is called as staggered cut or cut that makes sticky ends. Any complimentary sequence to this sequence will form hydrogen bond easily, therefore widely preferred in recombinant DNA technology than blunt ends. Then the second type of cut is causing blunt ends so this cut both strands at the same site generating flush ends as you see like this in the case of Alu1 so this is a recognition site agct so it will make cut at this position after g so this is after the cut so it will not produce any single standard extensions therefore the cut is called as blunt end or even cuts. The sticky ends are preferred in recombinant DNA technology as you see if the gene of interest is also having sticky ends and the vector is also having sticky ends, then it is very easy to anneal as these single stranded regions will form hydrogen bonds easily, that's why it is called as sticky ends. Now moving into ligases. Ligases are called the molecular glue in recombinant DNA technology as it seals or joins two DNA fragments. Ligases are joining enzymes that joins DNA fragments by forming phosphodiester bond. The process is called as ligation. These two DNA molecules are joined by ligases by means of phosphodiester bond formation. Now let us see how ligase enzyme joint DNA molecule. These are the nucleotides in a DNA strand as you see this is the first nucleotide second one and this is the third one, phosphodiester bond is formed between the five prime phosphate of one nucleotide of one fragment and three prime -OH of another fragment, this two ester bond called as phosphodiester bond is formed by enzyme ligase. Tt requires ATP or NAD plus for its activity example is T4 ligase. let me make it more clear, as you see this is a single stranded nick so there is a three three prime OH end and five prime phosphate region so in the presence of ATP this ligase enzyme joins and forming a phosphodiester bond just like this, thus joining the two DNA molecule or sealing the nick or single stranded break or nick. In short DNA ligase is the enzyme that forms that seals the nick or single stranded breaks by forming phosphodiester bond just like this.