Molecular BiologyXu Liyan

Chapter 15gene recombination and gene engineering

section 1section 1DNA Recombination

section 2section 2

Recombinant DNA technologyRecombinant DNA technology

section 3section 3

Relationship between Recombinant Relationship between Recombinant DNA technology and MedicineDNA technology and Medicine

section 1section 1DNA Recombination

1.1 Homologous Recombination

1.2 Gene Transfer and Recombine in Bacteria Conjugation Transformation Transduction

1.3 Site-specific Recombination

1.4 Transpositional Recombination

1.1 Homologous Recombination

The covalence connection between different

DNA moleculars is called DNA recombination

or gene recombination

The gene recombination includes two types

as follows

homologous recombination

site-specific recombination

transpositional recombination

Homologous Recombination The recombination between homologous sequence

is known as homologous recombination

5’

5’

3’

3’

3’

3’

5’5’

Rec BCD

Rec A

Rec A

Rec BCD

5’

5’

3’

3’

3’

3’

5’

5’Ligase

Holliday

Mechanism of Homologous Recombination

5’3’

3’ 5’

5’

3’5’

3’

5’3’

3’ 5’

5’

3’5’

3’

5’ 3’3’ 5’

5’

3’5’

3’

Ruv C Ruv C

Ligase

5’3’

3’ 5’

5’

3’

3’

5’

Ligase5’3’

3’ 5’

5’

3’5’

3’

5’

5’

3’

3’

3’

3’

5’

5’

Holliday

1 2

3

45 n

FF--FF++

1.2 Gene Transfer and Recombine in Bacteria Conjugation

F factor

Transformation

There is foreign DNA.

The phenotype of organisms is changed.

The changed Phenotype is passed down stably

DNADNA

Diplococcus pneumoniae Diplococcus pneumoniae with capsule is toxicity to humanwith capsule is toxicity to human

Diplococcus pneumoniae Diplococcus pneumoniae without capsule is untoxicity to human.without capsule is untoxicity to human.

transformation of diplococcus pneunonineA well-known experiment Avery, 1943

Transduction

When virus is released from infected

one cell and go to infect other cell, the

DNA fragment transfer from one cell to

other cell. This is called the transduction.

bacteria 1

phage

integration

some factor

bacteria 2

bacteria 1

phage

integration

some factor

bacteria 2

DNA fragment transformation between two bacterium

phage is carrier

1.3 site-specific recombination The integration catalyzed by integrase between

two site-specific DNA fragments is known as

site-specific recombination.

1.4 Transpositional recombination

the displacement of some gene in the geno

me by insertion sequence or transposons

1.4.1 insertion sequence and its mediated

gene transposition The length of typical insertion sequence is

about 750~1500bp.

Typical insertion sequence includes two 9 ~ 41bp

inverted repeat sequence and a transposase.

A 4 ~12bp positive repeat sequence always link

to flanking of inverted repeat sequence.

Gene transposition by insertion sequence:

conservative transposition

duplication transposition

targettargettransposase

insertion sequences

targettarget

3’

3’

5’

transposase

5’

3’3’

transposasetransposase

transposase3’

5’

5’transposase

transposase

transposase

polymerase Iligase

transposase

transposase

transposase

duplication transposition

11

22

3’

1.4.2 structure of transposons

The transposon is a dispersive and repeat sequence

in the genome.

The transposon can transfer from one region to other

region of the genome.

The structure of transposon is similar to one of

insertion sequence.

The both insertion sequence and transposon contain

transposase gene and flanking inverted repeat

sequences, but transposon also contain a few other

genes.

The insertion sequence is the most simple transposon

in fact.

IS 10L

amp-R generepressor gene

transposase

insertion sequence

transposase gene

Tn3

tet-R geneIS 10L

Tn10

section 2section 2DNA recombination technologyDNA recombination technology2.1 the basic 2.1 the basic conceptconcept related with DNA related with DNA

recombination technologyrecombination technology

2.2 the basic 2.2 the basic principleprinciple of DNA of DNA

recombination technologyrecombination technology

2.1 the concept related with DNA recombination technology

2.1.1 DNA cloning

2.1.2 tool enzyme

2.1.3 target gene

2.1.4 gene vector

2.1.1 DNA cloning It is a process of DNA molecular amplification.

Usually, the first a target DNA fragment is

inserted to a vector and a recombinant (replicon)

is constructed.

The second the recombinant is transformed into

host cell and screen out the cell containing the

recombinant.

The last that cell is amplified, namely a mass of

target DNA molecule is gained.

2.1.2 tool enzyme

restriction endonuclease

DNA ligase

DNA polymerase I

reverse transcriptase

polynucleotide kinase

end-transferase

alkaline phosphatase

structural character of cutting site recognized by restriction enzyme

restriction endonuclease recognized sequence and cut

Bam H I Eco R I

GAATTCCTTAAG

GGATCCCCTAGG

5’

Pvu I Sst I

GAGCTCCTCGAG

CGATCGGCTAGC

5’

Alu I Sma I

AGCTTCGA

CCCGGGGGGCCC

5’5’

5’5’

5’

5’5’

5’

5’5’

2.1.3 target gene

The interested gene is the target gene

source of the target gene

* It is from genomic DNA directly, this is

prokaryotic gene only generally.

* It is from artificial synthesis, this is simple

polypeptide gene generally.

* It is from mRNA.

* It is from genomic library or cDNA library.

* Polymerase Chain Reaction (PCR).

synthesize cDNA from mRNA

AAA…AAA5’ 3’

mRNAAAA…AAA

3’5’

AAA…AAATTT...TTT

5’3’

5’ 3’

primer: oligo dTreverse

transcriptase

TTT...TTT5’3’basic hydrolysis

TTT...TTT5’

3’

?

TTT...TTT5’

3’AAA…AAA

DNA polymerase I

TTT...TTT5’

3’AAA…AAA

3’

5’

S1 nuclease

genomic library

genomic DNA fragment 50-200kb

extraction

restrictively cut

gene fragments

recombination

recombinant

transformation

genomic library

target gene

cDNA library

recombination

recombinant

transformation

cDNA library

extractionextraction

mRNA

cDNAdoublestrands

5' 3'

extension

3' 5'

PCR Process

5' 3'

5' 3' 3' 5'

denaturation

annealing

3' 5'

5' 3'5'3'

3' 5'5' 3'

5'

5'

5' 3'

3' 5'

denaturation Next cycle

5' 3'

5'3'

2.1.4 gene vector

The gene vectors are DNA molecules,

which structure is reconstructed.

They can carry target DNA fragment

The target gene or DNA fragment is amplified

and expressed.

vector

* plasmid

* cosmid

* phage

* M13 phage

* insect virus DNA (autograph californica virus , ACNPV)

* yeast artificial chromosome DNA

* vaccinia virus DNA

* simian virus 40 DNA

3-10kb

40kb

29-48.5kb

5.243kb

180kb

6.407kb

20kb

4-8kb

15kb

0.3-1.0kb

2.5kb

128kb100kb

25kb

* bovine papilloma virus DNA

8.0kb 10kb

0.2-2.2Mb0.3-1.2Mb

* retrovirus DNA

* fowlpox virus DNA

* adenovirus DNA

* herpes simplex virus DNA

* cytomegalovirus DNA

* Epstein-Barr virus DNA

240kb

170kb

6.407kb

233-238kb

8-10kb

24-36kb

Xmn I 3966

2034 Xmn I

Pst I 3612

2067 Pvu II

1424 Ava I

650 Sal I

375 BamH I

plasmidplasmid

pBR3224.36kb

29 Hind III EcoR I 0

A origin

A screening gene

A single restriction site

condition

tetrampr

ori

plasmid

pUC192.69kb

Eco R I

Sac I

Kpn I

Sma I

Bam H I

Xba I

Hinc II

Pst I

Sph I

Hind III

ampr

ori

polylinker

52bpPlac

lac I

lac Z’

2.2 the basic principle of DNA2.2 the basic principle of DNA

recombination technologyrecombination technology

the procedureof gene cloning

separate targetgene as well as

vector11

cut target gene and vector

restrictedly22

join targetgene and vector33

recombinanttransformation44

separate targetgene as well as

vector11

cut target gene and vector

restrictedly22

ligate targetgene and vector33

recombinantscreening55

recombinantscreening55

recombinanttransformation44

recombinantscreening55

go a step further...

target geneamplify66

incomplete

1 2 31+3

incomplete digestion

1

2

3

Sma Icomplete

1+2+3

1 ------ 2, 22 ------ 5, 33 ------ 9, 44 ------ 14, 556

n n+n(n+1)/2 n+1

separate target gene

cut and ligate target gene and vector

CGGC

CGGC

Hpa II

CCGGGGCC

CCGGGGCC

Hpa IIgenome DNA

CGGC

CGGC

Hpa II

CCG

GG

GCC

CCGGGGCC

Ligase

recombinantrecombinant

CCGG

GGCC

plasmid

Hpa II

recombinant transformation

vectors and recombinants

competent cells cells

recombinant screening

amp or tet etc

++

plasmid plasmid extractionextraction

digist with digist with restriction

enzeme

11

22

11

22

marker

- +11

22

marker

- +

target gene amplification

食管癌细胞 NGAL 基因 5’ 端转录调控区不同长度片段 PCR 扩增结果

200bp

M 1431 1137 945 657 416 152 1124

2000bp

1000bp

重组子 pGEM-1431~152 XhoI 和 BglII 双酶切后，琼脂糖凝胶电泳鉴定结果

200bp

M1 1431 1137 945 657 416 152 1124 M2

1000bp ← 947 bp

← 5.0 kb

← 2.0 kb

重组子 pGLP-1431~152 XhoI 和 BglII 双酶切后，琼脂糖凝胶电泳鉴定结果

M 1431 1137 945 657 416 152

5000bp

1375 bp

564bp

target gene expression

prokaryotic expression system

D

Expression analysis of four expression vectors in E.coli by SDS-PAGE

eukaryotic expression system

21kDa → ← 25kDa

1 2 3 4 5 6 7 8 9 10

section 3section 3

the the relationship between DNAbetween DNA

recombination technology and medicinerecombination technology and medicine

discover and separate pathogenic gene

biopharmacy

DNA diagnosis

gene therapy

prevent transmissibility disease

SummarySummary

Homologous RecombinationSite-specific Recombination

Transposition Conjugation Transformation Transduction

DNA cloning: separate , cut, ligate ,

transform, screen, amplify,

express

选择题练习基因重组与基因工程

1. 基因工程的特点是

A 在分子水平上操作 , 在分子水平上表达

B 在分子水平上操作 , 在细胞水平上表达

C 在细胞水平上操作 , 在分子水平上表达

D 在细胞水平上操作 , 在细胞水平上表达

E 以上均可以

2. 限制性核酸内切酶不具有哪项特点 ?

A 仅存在于原核细胞中

B 用于重组 DNA 技术中的位 I 类酶

C 能识别双链 DNA 中特定的碱基顺序

D 具有一定的外切酶活性

E 辨认得核苷酸序列常具有回文结构

3. 有关质粒的叙述 , 下列哪项是错误的 ?

A 小型环状双链 DNA 分子

B 可小到 2-3kb, 大到数百个 kb

C 能在宿主细胞中独立自主地进行复制

D 常含有耐药基因

E 只有一种限制性核酸内切酶切口

4. 下列哪项不是重组 DNA 的连接方式 ?

A 粘性末端与粘性末端的连接

B 平端与平端的连接

C 粘性末端与平端的连接

D 人工接头连接

E 同聚物加尾连接

5. DNA 克隆不包括下列哪项步骤 ?

A. 选择一个适合的载体

B. 限制性核酸内切酶在特异位点裂解质粒和目的基因

C. 用连接酶连接载体 DNA 与目的 DNA,形成重组体

D. 用载体的相应抗生素抗性筛选含重组体的细菌

E. 重组体用融合法导入细胞

6. 下列哪种酶是重组 DNA 技术中最重要的 ?

A 反转录酶

B 碱性磷酸酶

C 末端转移酶

D DNA 聚合酶 I

E DNA 连接酶

7. 基因工程中通常使用的质粒存在于

A 细菌染色体

B 酵母染色体

C 细菌染色体外

D 酵母染色体外

E 以上均不是

8. 在已知 DNA 序列情况下 , 获取目的 DNA 最方便的方法是

A 人工化学合成

B 基因组文库法

C cDNA 文库法

D PCR 法

E 从染色体 DNA 直接提取

9. 基因工程中使目的基因与载体拼接的酶是

A DNA 聚合酶

B RNA 聚合酶

C DNA 连接酶

D RNA 连接酶

E 限制性核酸内切酶

10. 表达人类蛋白质的最理想的细胞体系是

A E.coli 表达体系

B 原核表达体系

C 酵母表达体系

D 昆虫表达体系

E 哺乳类细胞表达体系

11. The 11. The nucleotide number which restriction enzyme recognize in DNA nucleotide sequence is

A 4, 5 or 6

B 5, 6 or 7

C 6, 7 or 8

D 4, 6 or 8

E 4 - 8

12. The technique used in identification of DNA is

A northern blotting

B southern blotting

C Western blotting

D affinity chromatography

E ion exchange chromatography

13. The way of gene recombination doesn’t include

A transformation

B transduction

C transposition

D change-over 转换

E integration

14. The abbreviation of polymerase chain reaction is

A PRC

B PER

C PDR

D BCR

E PCR

15. 对于重组体的筛选 , 属于非直接选择法的是

A 免疫化学法

B 原位杂交法

C southern 印迹

D 补救标志筛选

E 酶联免疫筛选

16. 基因工程中 , 目的基因的来源有

A 化学合成

B PCR 合成

C cDNA 文库

D 基因组文库

E 组织细胞中染色体 DNA 直接提取

17. 质粒 DNA 等作为基因工程载体必须具备的条件是

A 能独立自主复制

B 易转化

C 易筛选 ( 质粒 DNA 含有抗药性基因等 )

D 具有合适的限制性核酸内切酶酶切位点

E 易提取获得

18. 将表达载体导入真核细胞的转染方法有

A 磷酸钙转染

B DEAE 葡萄糖介导转染

C 电穿孔

D 脂质体转染

E 显微注射

19. gene cloning also be called

A DNA recombination

B RNA recombination

C DNA cloning

D RNA cloning

E protein replication

20. The enzyme tools commonly used in gene cloning technique are

A restriction enzyme

B DNA polymerase I

C DNA ligase

D reverse transcriptase

E terminal transferase

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