Chapter 7 Genetic Manipulation 第七章遗传操作 DNA manipulation DNA的遗传操作 o Basic techniques used to clone genes Construction and screening of genomic libraries The manipulation of cloned DNa sequences in vitro The molecular analysis of DNA Cell manipulation细胞的遗传操作 ◆ Human gene therapy ◆ Transgenic animals
Chapter 7 Genetic Manipulation 第七章 遗传操作 ◼ DNA manipulation DNA的遗传操作 ◆ Basic techniques used to clone genes ◆ Construction and screening of genomic libraries ◆ The manipulation of cloned DNA sequences in vitro ◆ The molecular analysis of DNA ◼ Cell manipulation 细胞的遗传操作 ◆ Human gene therapy ◆ Transgenic animals
DNA manipulation DNA的遗传操作 Recombinant DNA techniques 到的基的gn9o1n现 目的基因与载体结合成重组DIA 重组DNA分子引入受体细胞并表达 转化体细胞的扩增、鉴定与筛选
DNA manipulation DNA的遗传操作 Recombinant DNA techniques 目的基因(the gene of interest)的获取 目的基因与载体结合成重组DNA 重组DNA分子引入受体细胞并表达 转化体细胞的扩增、鉴定与筛选
Basic techniques used to clone genes The discovery of restriction endonucleases 限制性核酸内切酶 The production of recombinant DNA molecules in vitro Amplification of recombinant DNA molecules in cloning vectors
Basic techniques used to clone genes ✓ The discovery of restriction endonucleases 限制性核酸内切酶 ✓ The production of recombinant DNA molecules in vitro ✓ Amplification of recombinant DNA molecules in cloning vectors
Restriction endonucleases限制性核酸内切酶 限制与修饰作用 E coli K EOP=10-4 E OP=1 EOP=k 入K E coli B B 限制性核酸内切酶 restriction endonucleases
Restriction endonucleases 限制性核酸内切酶 ▪ 限制与修饰作用 ▪ 限制性核酸内切酶restriction endonucleases E.coli K E.coli B E.O.P = 1 E.O.P = 10-4 E.O.P = 1 λ .B λ .K
限制酶一能识别和切断外来的 DNA而使它失去复制能力 修饰酶一能使限制酶的识别序 列中的若干个碱基甲基化,限制酶 不能识别它
限制酶—能识别和切断外来的 DNA而使它失去复制能力 修饰酶—能使限制酶的识别序 列中的若干个碱基甲基化，限制酶 不能识别它
EcoRI cleavage sites DNA troT GAATTU 9AA丁了C DNA from Species I CTIAAG species 2 Digest with restrictio endonuclease EcoRI mT 3 ECoRI fragments CIAA TAA with complementary 53 5’ single-stranded ends Mix digested DNAS and incubate under annealing conditions. 5'f 3 Base-pairing between the complementary single-stranded ends of cleaved DNA molecules Treat annealed DNA fragments with DNA ligase DNA from iA DNA trom Species 1 3 Species 2 EcoRI cleavage site Recombinant DNA molecules
Recognition Sequences and Cleavage Sites of Representative Restriction Endonucleases NuImber of Recogmition Sequences per Recognition Sequence Chromosone o Enzie source an Cleavage Sites Plinge A sv40 Virus EcORI Escherichia coli AA TTO CTT AAG Hindi Herophilus influenzae GTPV PUACc CAPu PyTG HindIll Hemophilus influenzae AAG CTT TIC GAA p Hemophilus parainfluenzae GTT AAC CAA TTO Hpall Hemophilus parainfluenzae 750 GG CC
Features of restriction endonucleases 0限制性核酸内切酶可分型,型和I型三类 o识别序列有3个特点:由46个核苷酸组成, 回文结构,可被有规律替代 oI型限制酶的切割类型:错切和平切两类
Features of Restriction endonucleases o 限制性核酸内切酶可分I型, II型和III型三类 o 识别序列有3个特点：由4~6个核苷酸组成， 回文结构，可被有规律替代 o II型限制酶的切割类型： 错切和平切两类
Amplification of recombinant DNA molecules in cloning vectors niou The essential features EcoRI cleavage site of a cloning vector 5 GAATTC 3 3'CTTAAG 5 Imp Dominant selectable marker Origin of gene conferring resistance to replication ampicillin to the host bacterium
Amplification of recombinant DNA molecules in cloning vectors The essential features of a cloning vector
(1 Digest with restriction endonuclease EcoRI Central portion is not required A left arm for replication or packaging 2. right arm Strategy employed in (2)Isolate EcoRI fragment using phage n as a from species of interest. EcoRI fragment cloning vector from foreign DNA (3 Mix foreign EcoRI fragment with left and right ) DNA arms under annealing conditions 2, left arm Foreign DNA 入 right arm 入DNA Foreign DNA 入DNA Package recombinant DNA molecules in A phage particles in vitro. Phage A particle carrying a recombinant DNa molecule
Strategy employed in using phage as a cloning vector