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
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
Mechanism of Homologous Recombination
5’
3’
3’
5’ Rec BCD
3’
5’
5’
3’
Rec A
Rec A
Rec BCD
5’
3’
Ligase
3’
5’
Holliday
3’
5’
5’
3’
3’
3’
5’
5’
Holliday
5’
3’
3’
5’
3’
5’
3’
3’
5’
5’
5’
3’
5’
5’
3’
3’
5’
Ruv C
3’
5’
Ligase
3’
3’ 5’
Ruv C
3’
5’
3’
5’
5’
3’
Ligase
5’
3’
3’
5’
5’
3’
5’
3’
3’
5’
5’ 3’
5’
3’
5’ 3’
1.2 Gene Transfer and Recombine in Bacteria
Conjugation
F-
F+
1
2
F factor
3
n
5
4
Transformation

There is foreign DNA.

The phenotype of organisms is changed.

The changed Phenotype is passed down stably
transformation of diplococcus pneunonine
A well-known experiment Avery, 1943
DNA
Diplococcus pneumoniae without capsule is untoxicity to human.
Diplococcus pneumoniae with capsule is toxicity to human
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
 DNA fragment transformation
between two bacterium
 phage is carrier
some factor
bacteria 2
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
genome 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
duplication transposition
insertion sequences
transposase
5’
3’
5’ 3’
target
transposase
transposase
3’
transposase
3’
5’
transposase
transposase
polymerase I
ligase
3’
target
3’
5’
transposase
1
transposase
transposase
transposase
transposase
2
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.
insertion sequence
transposase
Tn3
transposase gene repressor gene amp-R gene
Tn10
IS 10L
tet-R gene
IS 10L
section 2
DNA recombination technology
2.1 the basic concept related with DNA
recombination technology
2.2 the basic principle of DNA
recombination 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
Alu I
Sma I
recognized sequence and cut
5’
5’
AGCT
TCGA
5’
CCCGGG
GGGCCC
5’
GGATCC
CCTAGG
5’
Bam H I
5’
Eco R I
5’
GAATTC
CTTAAG
5’
5’
CGATCG
GCTAGC
5’
GAGCTC
CTCGAG
5’
Pvu I
Sst I
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
5’
5’
AAA…AAA3’
3’
TTT...TTT5’
AAA…AAA 3’
S1 nuclease
AAA…AAA3’
5’
mRNA
TTT...TTT5’
AAA…AAA
3’
DNA polymerase I
3’
reverse
transcriptase primer: oligo dT
5’
3’
TTT...TTT5’
AAA…AAA 3’
TTT...TTT
basic hydrolysis
?
5’
3’
TTT...TTT5’
genomic library
cDNA library
target gene
extraction
extraction
genomic DNA
fragment
50-200kb
mRNA
restrictively cut
cDNA
double
strands
gene fragments
recombination
recombination
recombinant
transformation
genomic library
recombinant
transformation
cDNA library
5'
3'
3'
5'
denaturation
5'
3'
3'
5'
annealing
5'
3'
3'
5'
3'
5'
3'
5'
extension
5'
5'
3'
3'
5'
5'
denaturation
5'
3'
Next cycle
3'
3'
5'
5'
5'
3'
PCR Process
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
3-10kb
15kb
*  phage 29-48.5kb 20kb
* cosmid
4-8kb
* insect virus DNA
(autograph californica
virus , ACNPV)
128kb
100kb
40kb
* M13 phage 6.407kb 0.3-1.0kb
* fowlpox virus DNA 6.407kb
* simian virus 5.243kb 2.5kb
40 DNA
* retrovirus DNA 8-10kb
* bovine papilloma 8.0kb 10kb
virus DNA
* adenovirus DNA 24-36kb
* Epstein-Barr virus DNA
170kb
* yeast artificial 0.2-2.2Mb
chromosome DNA 0.3-1.2Mb
* herpes simplex virus DNA 233-238kb
* vaccinia virus DNA 180kb 25kb
* cytomegalovirus DNA
240kb
plasmid
Xmn I 3966
EcoR I 0 29 Hind III
375 BamH I
Pst I 3612
650 Sal I
pBR322
4.36kb
condition
1424 Ava I
A origin
A screening gene
A single restriction site
2034 Xmn I
ori
2067 Pvu II
Eco R I
plasmid
Sac I
lac Z’
Kpn I
Sma I
Bam H I
Plac
pUC19
2.69kb
lac I
Xba I
Hinc II
Pst I
Sph I
Hind III
polylinker
52bp
2.2 the basic principle of DNA
recombination technology
the procedure
of gene cloning
go a step further...
separate target
1 gene as well as
vector
6
target gene
amplify
cut target
2 gene and vector
restrictedly
5
recombinant
screening
ligate
target
join target
3 gene and vector
4
recombinant
transformation
separate target gene
incomplete digestion
1
2
3
4
5
6
Sma I
complete
2
3
incomplete
1
2
3
4
5
n n+n(n+1)/2 n+1
1+2+3
1
------ 2,
------ 5,
------ 9,
------ 14,
2
3
1+3
cut and ligate target gene and vector
CCGG
Hpa II
GGCC
genome DNA
Hpa II
plasmid
CCGG
GGCC
CCGG
GGCC
Hpa II
CGG
C
C
GGC
Hpa II
CGG
C
C
GGC
Ligase
recombinant
recombinant transformation
vectors and recombinants
competent cells
recombinant screening
plasmid
extraction
amp or
tet etc
digist with
restriction
enzeme
-
-
+
1
1
2
2
marker
marker
1
+
+
2
target gene amplification
SHEEC genomic DNA
PCR amplification
cloned into pGEM-T easy vector
pGL3-promoter
pT-X
Recovered by
Minielute Gel
Extraction Kit
Xho I+ Bgl II
X target fragments
Dephosphorylation,Purification by
Quick PCR Purification Kit
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
Xho I+ Bgl II
Isolation recombinant plasmids by
QIAprep Miniprep Kit
pB-X recombinants
The agarose gel electrophoresis of PCR products of NGAL
gene 5’ flanking regulation sequences from SHEEC cells
M
2000bp 
1000bp 
200bp
1431 1137 945 657
416 152 1124
The agarose gel electrophoresis of recons pGEM1431~152 after XhoI + BglII digesting
M1 1431 1137 945 657 416
152 1124 M2
← 5.0 kb
← 2.0 kb
1000bp 
200bp
← 947 bp
The agarose gel electrophoresis of recons pGLP-1431~152
after XhoI + BglII digesting
M
5000bp 
1375 bp 
564bp 
1431
1137
945
657
416
152
target gene expression
prokaryotic expression system
D
Expression analysis of four expression vectors in E.coli
by SDS-PAGE
eukaryotic expression system
1
21kDa →
2
3
4
5
6
7
8
9 10
← 25kDa
section 3
the relationship between DNA
recombination technology and medicine

discover and separate pathogenic gene

biopharmacy

DNA diagnosis

gene therapy

prevent transmissibility disease
Summary
Homologous
Recombination
Site-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 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