AllrightsreservedExperiment 2Restriction Enzyme Digestion and Agarose GelElectrophoresisSchool of Life Sciences Tsinghua University
Experiment 2 Restriction Enzyme Digestion and Agarose Gel Electrophoresis All rights reserved
1. ObjecttivesLearn the characteristics of restrictionendonuclease and agarose gel electrophoresisto separate DNAs.?School of Life Sciences Tsinghua University
1. Objecttives • Learn the characteristics of restriction endonuclease and agarose gel electrophoresis to separate DNAs
2.Background. Restriction endonucleaseRestriction endonuclease cuts double-stranded DNAat specific restriction sites and produces restrictionfragments. They are called “tool enzymes", togetherwith DNA polymerase, DNA ligase, and terminaltransferase.It is an important tool in DNA recombination andalso widely used in studies of DNA primary structure.recombinant DNA technology and other fields ofmolecular genetics and molecular biologySchool of Life Sciences Tsinghua University
2. Background • Restriction endonuclease Restriction endonuclease cuts double-stranded DNA at specific restriction sites and produces restriction fragments. They are called “tool enzymes”, together with DNA polymerase, DNA ligase, and terminal transferase. It is an important tool in DNA recombination and also widely used in studies of DNA primary structure, recombinant DNA technology and other fields of molecular genetics and molecular biology
Key pointsHost-controlled restriction and modificationClassification and characteristics of restriction endonucleaseIsoschizomerEnzyme nomenclature of restriction endonuclease Factors that impact the activity of restriction endonuclease文School of Life Sciences Tsinghua University
① Host-controlled restriction and modification. ② Classification and characteristics of restriction endonuclease. ③ Isoschizomer. ④ Enzyme nomenclature of restriction endonuclease. ⑤ Factors that impact the activity of restriction endonuclease. Key points
Host-controlled restrictionand modificationRestriction and modification system (or restrictionmodification system, R-M system) is one of thedefensive mechanisms in bacterial cell. All of bacteriacan produce one or more kinds of restriction enzymesthat can incise double strain DNA. Cell controls the levelof extraneous DNA by these enzymes, but those DNAsthat codes these restriction enzymes are not influencedbecause other modification enzymes produced in this cellcan modify the DNA. Restriction enzymes can not incisethe modified DNA. This phenomenon is called host-controlled restriction and modification or host dependentrestriction and induced modificationSchool of Life Sciences Tsinghua University
Restriction and modification system (or restrictionmodification system, R-M system) is one of the defensive mechanisms in bacterial cell. All of bacteria can produce one or more kinds of restriction enzymes that can incise double strain DNA. Cell controls the level of extraneous DNA by these enzymes, but those DNAs that codes these restriction enzymes are not influenced because other modification enzymes produced in this cell can modify the DNA. Restriction enzymes can not incise the modified DNA. This phenomenon is called hostcontrolled restriction and modification or host dependent restriction and induced modification. Host-controlled restriction and modification
Classification and characteristics ofrestriction endonucleaseBased on the composition, structurecomplexity, cofactor requirements, substratespecificity and cleavage patterns , restrictionendonucleases are classified as three types:Type III *TypeIIITypeXSchool of Life Sciences Tsinghua University
Classification and characteristics of restriction endonuclease Based on the composition, structure complexity, cofactor requirements, substrate specificity and cleavage patterns , restriction endonucleases are classified as three types: Type Ⅰ Type Ⅱ* Type Ⅲ
Type I restriction endonuclease:Recognize specific nucleic acid sequence andincise double strain DNA around this specific site, butthe nucleic acid seguences incised by these enzymesdo not have specificity. The incision is random.Such as EcoB、 EcoK, they are not very useful inDNA recombination and genetic engineering. Theycan not be used to analyse the structure of DNA or toclone genes.文School of Life Sciences Tsinghua University
• Type I restriction endonuclease: Recognize specific nucleic acid sequence and incise double strain DNA around this specific site, but the nucleic acid sequences incised by these enzymes do not have specificity. The incision is random. Such as EcoB、EcoK, they are not very useful in DNA recombination and genetic engineering. They can not be used to analyse the structure of DNA or to clone genes
Type II restriction endonuclease:Recognize specific nucleic acid sequence and incise doublestrain DNA in specific sites in this sequence. With the specificrecognition and incision, type II restriction endonuclease isone of the most widely used enzyme tools in DNArecombination.Generally the nucleic acid sequence recognized by type Irestriction endonuclease contains 4 or 6 nucleosides. Somenucleic acid sequences also contain 5,7,8,9,10,11 nucleosidesThe nucleic acid sequence recognized by type II restrictionendonuclease is a kind of palindrome. There are two patternsof digestion for type II restriction endonuclease:School of Life Sciences Tsinghua University
• TypeⅡ restriction endonuclease: Recognize specific nucleic acid sequence and incise double strain DNA in specific sites in this sequence. With the specific recognition and incision, typeⅡ restriction endonuclease is one of the most widely used enzyme tools in DNA recombination. Generally the nucleic acid sequence recognized by typeⅡ restriction endonuclease contains 4 or 6 nucleosides. Some nucleic acid sequences also contain 5,7,8,9,10,11 nucleosides. The nucleic acid sequence recognized by typeⅡ restriction endonuclease is a kind of palindrome. There are two patterns of digestion for typeⅡ restriction endonuclease:
Cohesive end (Sticky end):For example: nucleic acid sequence recognized by EcoRl:5'...... G'AATT C......3'3'...... C TTIAA'G...... 5After digestionAATTC......35.......GG......5'3'......CTTAASchool of Life Sciences Tsinghua University
Cohesive end (Sticky end): For example: nucleic acid sequence recognized by EcoRI:
Blunt end:For example: nucleic acid sequence recognized by EcoRV5... GAT"ATC...... 3'3'...... CTA'TAG...... 5"AfterdigestionATC...... 3'... GAT5'..3'...... CTATAG......5'XSchool of Life Sciences Tsinghua University
Blunt end: For example: nucleic acid sequence recognized by EcoRV: