Analysis and Characterization of Restriction enzymes (RE)

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Presentation transcript:

Analysis and Characterization of Restriction enzymes (RE) Types of REs Mapping of restriction enzyme sites

Restriction Enzymes Type I Type II Type III Methylation/cleavage (3 subunits) >1000 bp from binding site e.g., Eco AI GAGNNNNNNNGTCA Type II Cleavage at specific recognition sites Type III Methylation/cleavage (2 subunits) 24–26 bp from binding site e.g., Hinf III CGAAT

Restriction Endonucleases: Type II Enzyme Isolated from Recognition sequence Eco RI E. coli, strain R, 1st enzyme Gν AATTC Eco RV 5th enzyme Gv ATATC Hind III H. influenzae, strain d, 3rd enzyme Av AGCTT

Restriction Enzymes BamH1 HaeIII KpnI Cohesive Ends Blunt Ends GGATCC CCTAGG HaeIII GGCC CCGG Cohesive Ends (5´ Overhang) (3´ Overhang) KpnI GGTACC CCATGG Blunt Ends (No Overhang)

Restriction Enzymes GATC CTAG GGCC CCGG CCCGGG GGGCCC GGATCC CCTAGG DpnI (Requires methylation) Methylation-sensitive Enzymes GGCC CCGG HaeIII (Inhibited by methylation) CCCGGG GGGCCC XmaI (5’ Overhang) SmaI (Blunt Ends) Isoschizomers Enzymes Generating Compatible Cohesive Ends GGATCC CCTAGG BamHI AGATCT TCTAGA BglII CTCGTG GAGCAG BssSI NNCAGTGNN NNGTCACNN TspRI (3’ Overhang) Enzymes Recognizing Non palindromic Sequences

Ligation of Restriction Enzyme Digested DNA Sticky ends must match (be complementary) for optimal re-ligation. Sticky ends can be converted to blunt ends with nuclease or polymerase. Blunt ends can be converted to sticky ends by ligating to synthetic adaptors. Blunt ends can be re-ligated with less efficiency than sticky ends.

Restriction Enzyme Mapping Digest DNA with a restriction enzyme. Resolve the fragments by gel electrophoresis. The number of bands indicates the number of restriction sites. The size of the bands indicates the distance between restriction sites.

Restriction Enzyme Mapping 4.3 kb 3.7 kb 2.3 kb 1.9 kb 1.4 kb 1.3 kb 0.7 kb BamH1 XhoI 4.0 kb 2.8 kb 1.2 kb 1.7 kb 1.1 kb BamH1 XhoI 1.1 kb 1.7 kb 1.2 kb 2.8 kb

Summary Restriction enzymes cut DNA at specific recognition sequences. DNA can be characterized by restriction enzyme mapping.