lecture25 - Type II Restriction Enzymes Recognition...

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Type II Restriction Enzymes Some generate sticky ends ex. Bam HI, Eco RI, ClaI, Hind III, Not I, Pst I, Tth lllI --Some sticky ends are 5’ overhangs ex. Eco RI, Cla I, Hind III --Some sticky ends are 3’ overhangs ex. Pst I, Tth 111I Some generate blunt ends ex. Hae III, PvuII, Sma I Recognition sequences for some type II restriction enzymes Bam H1 GGATCC CCTAGG Eco RI GAATTC CTTAAG Pst I CTGCAG GACGTC Pvu II CCCGGG GGGCCC
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Types of Sticky Ends 5’ overhangs--ex. Eco R1 ---GAATTC-- ---CTTAAG-- 5’---G AATT C---3’ 3’---C TTAA G---5’ + 3’ overhangs--ex. Pst I ---CTGCAG-- ---GACGTC-- 5’--C TGCA G--3’ 3’--G ACGT C--5’ +
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Compatibility of Sticky Ends to Re-Ligate Overhangs cut with same enzyme can be religated using DNA ligase (will regenerate same restriction site) Some overhangs cut with different enzymes are complementary and can be ligated (may or may not regenerate site) Most overhangs cut with different enzymes are not complementary ex. Eco R1 Bam HI Bgl II ---GAATTC-- --GGATCC-- --AGATCT-- ---CTTAAG-- --CCTAGG-- --TCTAGA--
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How to Make Blunt Ends Out of Sticky Ends + For 5’ Overhangs--need to fill-in with DNA polymerase Eco RI --G --AATTC-- --GAATT AATTC-- --CTTAA G-- --CTTAA TTAAG-- Klenow fragment of DNA Pol I dNTPs For 3’ Overhangs--need to chew back with exonuclease Pst I --CTGCA G-- --C G-- --G ACGTC-- --G C-- + T4 DNA polymerase or mung bean nuclease + dNTPs 5’ 5’ 3’ 3’ 3’ 3’ 5’ 5’ 5’ 5’ 5’ 5’ 3’ 3’ 3’ 3’
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lecture25 - Type II Restriction Enzymes Recognition...

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