Tuition Center Singapore JC H2 H1 Biology Tuition – Basic Tools in Genetic Engineering

Basic Tools in Genetic Engineering

Key Technique 1 : Restriction enzymes (endonucleases) 

• Restriction enzymes are synthesized naturally by ribosomes in bacteria.
• Different species of bacteria synthesize specific types of restriction enzymes which protect them from viruses by degrading incoming viral DNA.
• The bacterial genome is protected from degradation by its own restriction enzyme
• by addition of a methyl group (-CH3) to an adenine (A) or cytosine (C) at the restriction site, whereas
• the DNA sequence of an invading virus is generally not methylated.
• Each restriction enzyme (endonuclease) recognizes a specific sequence of 4 to 8 nucleotide bases on the DNA molecule known as restriction site. Restriction sites are palindromic
• A palindromic sequence reads the same on one strand in a 5’ to 3’ direction as that on the other strand in a 5’ to 3’ direction 
• The restriction enzyme acts on the restriction site by breaking the phosphodiester bond at a position between 2 specific nucleotides.
• It acts like a molecular scissors, cutting up DNA molecules into fragments called restriction fragments.

• Restriction enzymes produce restriction fragments with Sticky ends (sometimes called ‘cohesive ends’)         

              >Restriction enzymes cut covalent phosphodiester bonds of both DNA strands, often in a staggered                 way creating single–stranded ends called sticky ends.

              > These single-stranded ends will form hydrogen-bonded base pairs with complementary single-stranded stretches on the other DNA molecule cut with the same restriction enzyme.

• Restriction enzymes produce restriction fragments with Blunt ends

              >Some restriction enzymes make a simple cut across both strands at a single point, forming blunt ends.

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