EcoRI sites( +) A C D E F G (The largest fragment) (The smallest fragment) Consider a linear dna E molecule with 6 copies B of Aattc: A it will be cut into 7 decreasing size fragments which could D be separated in the gel electrophoresis by size Fig 20-3 digestionof a DNA fragment with endonuclease ECORI
16 ◼ Consider a linear DNA molecule with 6 copies of GAATTC: it will be cut into 7 fragments which could be separated in the gel electrophoresis by size (The largest fragment) (The smallest fragment) Fig 20-3 digestionof a DNA fragment with endonuclease EcoRI
Restriction digestion Endonucleases are used to make restriction map e.g. the combination of EcorI t Hindill AL e to be isolate and a given molecule to be identified aa given molecule will generate a characteristic series of patterns when digested with a set of different enzymes 17
17 ◼ Endonucleases are used to make restriction map: ◼ e.g. the combination of EcoRI + HindIII ◼ Allows different regions of one molecule to be isolate and a given molecule to be identified ◼ A given molecule will generate a characteristic series of patterns when digested with a set of different enzymes Restriction digestion
Restriction digestion Different enzymes recognize their specific target sites with different frequency a ECoRI Recognize hexameric sequence : 4-6 a Sau3al Recognize tetrameric sequence 4 4 Thus sau3a1 cuts the same dna molecule more frequently 18
18 Different enzymes recognize their specific target sites with different frequency ◼ EcoRI Recognize hexameric sequence: 4-6 ◼ Sau3A1 Recognize terameric sequence: 4- 4 ◼ Thus Sau3A1 cuts the same DNA molecule more frequently Restriction digestion
Restriction digestion Hpal 50 G T DOA A C 3 blunt ends 3'O C A AIOT T GI 5 cut ECORI 5 3 5′ cut HindIll sticky ends 5 AOA G C 3 3 C G AOA 5 c PstI 5 G C AlOG 3 5 C Fig 20-4 recognition sequences and cut sites of various endonucleases
19 sticky ends blunt ends Fig 20-4 recognition sequences and cut sites of various endonucleases Restriction digestion
Restriction digestion a The 5' protruding ends of are said to be sticky"because they readily anneal through base-pairing to DNA mnleriles rit With the same enzyme 50FGIOAATT C 3 A AOG 5′ cut Reanneal with its complementary strand or other sticky"end 6A A strands with the same cut AA-“ sticky"end Copyright 2004 Pearson Education, Inc publishing as Benjamin Cummings
20 ◼ The 5’ protruding ends of are said to be “sticky” because they readily anneal through base-pairing to DNA molecules cut with the same enzyme Reanneal with its complementary strand or other strands with the same cut Restriction digestion