Restriction endonucleases(Re)are the nucleases that cleave dna at particular sites by the recognition of specific sequences. a RE used in molecular biology typically recognize(识别) short(4-8bp) target sequences that are usually palindromic (回文结构), and cut(切割) at a defined sequence within those sequences eg.EcoR工 5. GAATTCI. 3 CTTAAG
17 ◼ Restriction endonucleases (RE) are the nucleases that cleave DNA at particular sites by the recognition of specific sequences. ◼ RE used in molecular biology typically recognize (识别) short (4-8bp) target sequences that are usually palindromic (回文结构), and cut (切割) at a defined sequence within those sequences. e.g. EcoRI 5’….GAATTC.….3’ ….CTTAAG…
How to name a restriction endonuclease EcoR工 Escherichia coli R13 the 1st such Species category strain enzyme found 18
18 the 1st such enzyme found Escherichia coli Species category R13 strain How to name a restriction endonuclease? EcoRI
How to estimate the frequency of the re in a dna molecule or genome? The random occurrence of the hexameric(六核苷酸的) sequence 1/4096(4-6) What are the frequencies if the recognition sequences are four (tetrameric)and eight(octameric) nucleotides? [homework 19
19 The random occurrence of the hexameric (六核苷酸的) sequence: 1/4096 (4-6 ) What are the frequencies if the recognition sequences are four (tetrameric) and eight (octameric) nucleotides? [homework] How to estimate the frequency of the RE in a DNA molecule or genome?
EcoRI sites() A B C D E F G (The largest fragment) (The smallest fragment) Consider a linear dna E molecule with 6 copies of GAATTC: A it will be cut into 7 decreasing size fragments which could be separated by the le gel electrophoresis F Fig 20-3 digestionof a DNA fragment with endonuclease ECORI
20 ◼ Consider a linear DNA molecule with 6 copies of GAATTC: it will be cut into 7 fragments which could be separated by the gel electrophoresis. (The largest fragment) (The smallest fragment) Fig 20-3 digestionof a DNA fragment with endonuclease EcoRI
Use of multiple REs allows different regions of a DNA molecule to be isolated A given molecule will generate a characteristic series of pattern when digested with a set of different enzymes e.g. the combination of EcoRI+ Hindiii 21
21 ◼ A given molecule will generate a characteristic series of pattern when digested with a set of different enzymes. ◼ e.g. the combination of EcoRI + HindIII Use of multiple REs allows different regions of a DNA molecule to be isolated