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Molecular Biology Xu Liyan. Chapter 14 gene recombination and gene engineering. section 1 DNA Recombination section 2 Recombinant DNA technology section 3 Relationship between Recombinant DNA technology and Medicine. section 1 DNA Recombination. 1.1 Homologous Recombination - PowerPoint PPT Presentation
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Molecular BiologyXu Liyan
Chapter 14gene 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
SHEEC genomic DNA PCR amplification
cloned into pGEM-T easy vector
pT-X pGL3-promoter
Recovered by Minielute Gel Extraction Kit
Xho I+ Bgl II Dephosphorylation,Purification by Quick PCR Purification Kit
X target fragments pGL3-promoter dephosphorylated vector Ligation by T4 Ligase
Transformed into JM109 competent cells
plated on LB plates containing ampicillin, cultured at 37 for 16 h ℃
pB-X cell clones
Isolation recombinant plasmids by
QIAprep Miniprep Kit
Xho I+ Bgl II
pB-X recombinants
The agarose gel electrophoresis of PCR products of NGAL gene 5’ flanking regulation sequences from SHEEC cells
200bp
M 1431 1137 945 657 416 152 1124
2000bp
1000bp
The agarose gel electrophoresis of recons pGEM-1431~152 after XhoI + BglII digesting
200bp
M1 1431 1137 945 657 416 152 1124 M2
1000bp ← 947 bp
← 5.0 kb
← 2.0 kb
The agarose gel electrophoresis of recons pGLP-1431~152 after XhoI + BglII digesting
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