DNA Recombination and Recombinant DNA technology

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1 DNA Recombination and Recombinant DNA technology
Chapter 21 DNA Recombination and Recombinant DNA technology

2 DNA recombination: different DNA molecules break and link to form new DNA molecules. recombinant DNA technology: two or more than two DNA molecules are combined together to form a new DNA molecules.

3 Section 1 DNA Recombination and Gene Transfer in Nature

4 DNA recombination homologous recombination site-specific recombination
transposition recombination conjugation transformation transduction

5 I. homologous recombination
The recombination happened between homologous sequences is called homologous recombination, also called general recombination.

6 Holliday model： 2 highly arranged homologous DNA molecules； 1 strand of 1 DNA molecules break , and linked with relative strand of the other DNA to form Holliday midbody. Holliday midbody separate to form heterogeneous double strands patch recombinant splice recombinant

7 endonulease endonulease DNA (recA) Branching (recA) (recBCD) ligase
5´ 3´ 5´ 3´ endonulease (recA) 5´ 3´ 3´ 5´ Branching (recA) endonulease (recBCD) 5´ 3´ DNA ligase 5´ 3´ Holliday midbody

8 Holliday endonuclease (ruvC) endonuclease (ruvC) DNA DNA 连接酶 连接酶 5´ 3´
splice recombinant patch recombinant

9 II. conjugation Plasmid DNA molecules are transferred from one cell to another while cell-cell or bacteria-bacteria contacting. plasmid Small circle double strand DNA molecules with self-replication function.

10 conjugation transfer

11 III. transformation ransformation
Exogenous DNA was obtained by cells and express new heredity phenotype.

12 eg：DNA fragment is absorbed by another bacteria while bacteriolysis.

13 IV. transduction Virus released from the host cell will infect another cell again, the DNA transfer happened between the donor cell and receptor cell is called transduction.

14 λ-phage bacteriolysis

15 Recombinant DNA Technology
Section 2 Recombinant DNA Technology

16 Also called gene engineering, means to repair or recombine genes and let the
living body generate new phenotype.

17 The history 1865, G.J.Mendel, the peas cross-breeding experiment.

18 E.Coli anti-streptomycin and anti-tetracycline
In 1973, the first recombinant DNA molecule was constructed in Stanford university. anti-streptomycin plasmid splicing Mosaic plasmid anti-tetracycline plasmid E.Coli E.Coli anti-streptomycin and anti-tetracycline

19 1997, cloned sheep “Dolly” in England

20 Some concepts about recombinant DNA technique:
DNA clone: clone: an aggregation of the same copies coming from one ancestor. cloning: the process that can obtain the same copies.

21 Technique level： molecular clone (DNA clone) cell clone
individual clone ( animal or plant)

22 molecular cloning, or DNA cloning or genetic engineering
Main process: to combine the purpose DNA fragment with vector to form a new recombinant DNA and to be replicated and amplified in receptor cell to obtain a large amount of copies of a gene.

23 purposes： ① to obtain a gene’s copy that we are interest in. ② to obtain the production of the gene---protein .

24 I. Common used tool enzymes in recombinant technology
restrictive endonuclease DNA polymeraseⅠ reverse transcription enzyme T4 DNA ligase alkaline phosphatase Taq DNA polymerase

25 restriction endonuclease
concept： restriction endonuclease, RE is a kind of nucleic endonuclease, which can recognize specific internal sequence and split phosphate dieser bond. Bam HⅠ GATCC G GGATCC CCTAGG G CCTAG +

26 HindⅢ classification： typeⅠ、typeⅡ、typeⅢ nomenclature：
Haemophilus influenzae d plant The third kinds of enzyme nomenclature： HindⅢ category series plant sequnce

27 typeⅡ —— palindrome GGATCC CCTAGG Cut edge：flat edge、sticky edge

28 + + Flat edge Sticky edge GTCGAC CAGCTG GGATCC CCTAGG GTC CAG GAC CTG
HindⅡ Bam HⅠ GTCGAC CAGCTG GGATCC CCTAGG GTC CAG GAC CTG GATCC G + + G CCTAG 左下角处有超级链接到配伍末端 Flat edge Sticky edge 28

29 isocaudarner Some restricted endonuclease recognize different palindrome sequence, but generate same sticky edge, the enzyme is called isocaudarner, the same end is called compatible end. Bam HⅠ Bg lⅡ GGATCC CCTAGG AGATCT TCTAGA A TCTAG GATCT A GATCC G + G CCTAG +

30 ：isoschizomers Restricted endonucleases coming from different resource，but recognize the same palindrome sequence. Bam HⅠ GGATCC CCTAGG G CCTAG GATCC G + BstⅠ GATCC G GGATCC CCTAGG G CCTAG +

31 restricted endonuclease
Name　　 　 palindrome Name 　　　 　palindrome BamHⅠ ’…G▼GATCC...3’ Bgl Ⅱ ’…A▼GATCT...3’ EcoR Ⅰ ’…G▼AATTC...3’ Hind Ⅲ ’…A▼AGCTT...3’ Hpa Ⅱ ’…C▼CGG...3’ Mbo Ⅰ ’…▼GATC...3’ Nde Ⅰ ’…GA▼TATG...3’ Apa Ⅰ ’…GGGCC▼C...3’ Hae Ⅱ ’…PuGCGC▼Py...3’ Kpn Ⅰ ’…GGTAC▼C...3’ Pst Ⅰ ’…CTGCA▼G...3’ Sph Ⅰ ’…GCATG▼C...3’ Alu Ⅰ ’…AG▼CT...3’ EcoR Ⅴ ’…GAT▼ATC...3’ Hae Ⅲ ’…GG▼CC...3’ Pvu Ⅱ ’…CAG▼CTG...3’ Sma Ⅰ ’…CCC▼GGG...3’

32 II. Common used vectors in recombinant technology
concept of vector: Some DNA molecules which can carry purpose gene we want to study and amplify the gene or express the protein encoded by the gene. classification of vectors: cloning vector expression vector

33 cloning vector The vectors designed for the insertion and amplification of exogenous DNA sequence. expression vector The vectors designed for the expression of the gene inserted in.

34 （一）克隆载体 1. 克隆载体应具备的基本特点 至少有一个复制起点使载体在宿主细胞中进行自主复制，并能使克隆的外源DNA片段得到同步扩增；
至少有一个选择标志（selection marker）：选择标志是区分含与不含载体的细胞所必需的，包括抗生素抗性基因、β-半乳糖苷酶基因（lacZ）、营养缺陷耐受基因等。 有适宜的RE的单一切点：载体中一般都构建有一段特异性核苷酸序列，在这段序列中包含了多个RE的单一切点，可供外源基因插入时选择，叫多克隆位点（multiple cloning sites，MCS）。

35 2. 常用的克隆载体 （1）质粒 (plasmid) 特点: 能在宿主细胞内独立自主复制；带有某些遗传信息, 会赋予宿主细胞一些遗传性状。

36

37 pUC18质粒载体图谱

38 （2）噬菌体(phage) λ噬菌体DNA改造系统 λgt系列（插入型，适用cDNA克隆） EMBL系列（置换型，适用基因组克隆）
M13噬菌体DNA改造系统（含lacZ基因） M13mp系列 pUC系列

39 （3）其他克隆载体 柯斯质粒（cosmid）载体（又称黏粒载体） 酵母人工染色体
(yeast artificial chromosome, YAC) 细菌人工染色体 (bacterial artificial chromosome, BAC) 动物病毒DNA改造的载体 （如腺病毒，腺病毒相关病毒，逆转录病毒）

40 （二）表达载体 表达载体是指用来在宿主细胞中表达外源基因的载体。 根据宿主细胞分为： 原核表达载体 真核表达载体

41 1. 原核表达载体 原核表达载体的基本组成 R：调节序列；P：启动子；SD：SD序列；TT：转录终止序列

42 OriPro：原核复制起始序列；P：启动子；MCS：多克隆位点；
2. 真核表达载体 真核表达载体的基本组成 OriPro：原核复制起始序列；P：启动子；MCS：多克隆位点； TT：转录终止序列；orieuk：真核复制起始序列。