Chapter 2 Enzymes used in GE Ø2.1 Restriction endonuclease Ø2.2 DNA ligase joining DNA molecules Ø2.3 DNA Polymerase and modifying enzymes Ø2.4 Nucleases 2.1 Restriction endonuclease • 1952年，Luria和Human，T偶数噬菌体 • 1953年，Bertani和Weigle，λ噬菌体 Restriction endonuclease: recognizes a short and specific DNA sequence and cuts it from inside. The specific DNA sequence is called recognition sequence cleavage site：cut site How restriction enzymes are named? Escherichia Coli Ry13 EcoR I 属名 种名 株系 编号 若种名头2个字母相同则其中一个可用种名的第一和第三个字母。 书写时前3个字母一定斜体，字母与罗马数字间要有空格。 Classification Recognition Sequences • Many restriction sequences are palindromic(回文,Read the same in the opposite direction ). For example, –5’ GAATTC 3’ –3’ CTTAAG 5’ • 回文诗： 春城一色柳垂新，色柳垂新自爱人 人爱自新垂柳色，新垂柳色一城春 Sticky End Cutters • Staggered cuts produce single stranded “sticky-ends” • A protruding, single-stranded nucleotide sequence produced when a restriction endonuclease cleaves off center in its recognition sequence. Blunt End Cutters • cutting across both strands of DNA. • These are called blunt end cutters AluI HaeIII isoschizomers.(同裂酶) • Restriction enzymes that have the same recognition sequence as well as the same cleavage site . Neoschizomers (新裂酶) • Some restriction enzymes cut the same recognition site but at different locations –Neoschizomers are a subset of isoschizomers. isocaudomers同尾酶 • Some restriction endonucleases produce the same nucleotide extensions but have different recognition sites complete digestion完全的酶切: cleave at all relevant restriction sites. partial digestion局部酶切: a restriction enzyme is controlled so that it cuts only some of the available sites. Storage • storage at –20°C for most restriction enzymes. • for periods longer than 30 days, storage at –70°C. • Glycerol(50%） is added to restriction enzyme storage buffers to prevent freezing at -20°C. • 10X Buffers and concentrated BSA should also be stored at –20°C. Digestion Assay restriction endonuclease activity: One unit of is defined the amount of enzyme required to completely digest 1 μg of substrate DNA ( λDNA） in a total reaction volume of 50 μl in an hour. Volume activity: U/μl.