Download different DNA molecules break and link to form

Chapter 21
DNA Recombination and
Recombinant DNA technology
• 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.
Section 1
DNA Recombination
and Gene Transfer in Nature
DNA
recombination
homologous recombination
site-specific recombination
transposition recombination
conjugation
transformation
transduction
I. homologous recombination
 The recombination happened between homologous
sequences is called homologous recombination, also
called general recombination.

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
5´
3´
3´
5´
5´
3´
5´ endonulease 3´
3´
5´
5´
5´
3´
3´
5´
3´
5´
5´
3´
3´
3´
5´
5´
3´ 5´
3´
(recA)
5´
Branching 3´
3´
(recA)
5´
3´
5´
5´
3´
5´
3´
3´
5´
3´
5´
5´
3´
endonulease
(recBCD)
5´
3´
3´
3´
3´
5´
5´
3´
DNA
ligase
Holliday midbody
5´
3´
3´
5´
3´
5´
5´
3´
3´ 5´
5´
3´
Holliday
endonuclease
(ruvC)
5´
3´
5´
3´
DNA
连接酶
3´
5´
3´
3´
5´
3´
5´
5´
3´
endonuclease
(ruvC)
3´
5´
3´
5´
3´ 5´
5´
3´
3´ 5´
patch recombinant
DNA
连接酶
3´5´
5´
3´
5´
5´3´
5´3´
5´
3´
3´
5´
splice recombinant
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.
conjugation
transfer
III. transformation
ransformation
Exogenous DNA was obtained
by cells and express new heredity
phenotype.
eg：DNA fragment is absorbed by another
bacteria while bacteriolysis.
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.
λ-phage
bacteriolysis
Section 2
Recombinant DNA
Technology
Also called gene
engineering, means
to repair or recombine
genes and let the
living body generate
new phenotype.
The history
1865, G.J.Mendel, the peas cross-breeding experiment.
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
1997, cloned sheep “Dolly” in England
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.
 Technique level：
 molecular clone (DNA clone)
 cell clone
 individual clone ( animal or plant)
 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.
 purposes：
① to obtain a gene’s copy that we are interest in.
② to obtain the production of the gene---protein .
I. Common used tool enzymes in
recombinant technology
 restrictive endonuclease
 DNA polymeraseⅠ
 reverse transcription enzyme
 T4 DNA ligase
 alkaline phosphatase
 Taq DNA polymerase
restriction endonuclease
 concept：
restriction endonuclease, RE is a kind of
nucleic e n d o n u c l e a s e , which can recognize
specific internal sequence and split phosphate
dieser bond.
Bam HⅠ
GGATCC
CCTAGG
GATCC
G
G
CCTAG +
 classification：
typeⅠ、typeⅡ、typeⅢ
 nomenclature：
Haemophilus influenzae d plant
The third kinds of enzyme
HindⅢ
category series plant
sequnce
 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
G
+ GATCC
CCTAG
G
A
+GATCT
A
TCTAG
typeⅡ —— palindrome
GGATCC
CCTAGG
Cut edge：flat edge、sticky edge
HindⅡ
GTCGAC
CAGCTG
GTC GAC
CAG + CTG
Flat edge
Bam HⅠ
GGATCC
CCTAGG
G
+ GATCC
G
CCTAG
Sticky edge
：isoschizomers
Restricted endonucleases coming from different
resource，but recognize the same palindrome sequence.
Bam HⅠ
GGATCC
CCTAGG
G
GATCC
+
CCTAG
G
GGATCC
CCTAGG
GATCC
G
+
G
CCTAG
BstⅠ
restricted endonuclease
Name
BamHⅠ
Bgl Ⅱ
EcoR Ⅰ
Hind Ⅲ
Hpa Ⅱ
Mbo Ⅰ
Nde Ⅰ
Apa Ⅰ
palindrome
Name
5’…G▼GATCC...3’
5’…A▼GATCT...3’
5’…G▼AATTC...3’
5’…A▼AGCTT...3’
5’…C▼CGG...3’
5’…▼GATC...3’
5’…GA▼TATG...3’
5’…GGGCC▼C...3’
Hae Ⅱ
Kpn Ⅰ
Pst Ⅰ
Sph Ⅰ
Alu Ⅰ
EcoR Ⅴ
Hae Ⅲ
Pvu Ⅱ
Sma Ⅰ
palindrome
5’…PuGCGC▼Py...3’
5’…GGTAC▼C...3’
5’…CTGCA▼G...3’
5’…GCATG▼C...3’
5’…AG▼CT...3’
5’…GAT▼ATC...3’
5’…GG▼CC...3’
5’…CAG▼CTG...3’
5’…CCC▼GGG...3’
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
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.
1. Clone vectors
Basic feature of clone vectors:
 At least 1 replication origin
 be able to self replicate in host cell and the exogenous
gene sequence can be amplified at the same time.
 At least 1 selection marker to distinguish the vectors
containing the exogenous gene
eg: antibiotics gene, β-lactase gene(lac Z)
 MCS（multiple cloning sites）a specific sequence in
vector containing multiple RE sequences.
Common used clone vectors
（1）plasmid
concept : small circle double strand DNA
molecules which can replicate themselves.
pUC18质粒载体图谱
（2）phage
 λ phage DNA editing system
λgt (suitable for cDNA cloning）
EMBL（ suitable for genomic DNA cloning ）
 M13 phage DNA editing system
M13mp series
pUC series
（3）other clone vectors
 cosmid
 yeast artificial chromosome, YAC
 bacterial artificial chromosome, BAC
 animal virus (adenovirus, retroviral vectors)
2. expression vectors
 Concept:
Means those vectors that can express the
exogenous gene in the host cells.
 Classification according to the host：
 eukaryotes expression vectors
 prokaryotes expression vectors
1. prokaryotes expression vectors
Transcription direction
Coding sequence
R：调节序列；P：启动子；SD：SD序列；TT：转录终止序列
integration sequence
2.eukaryotes expression vectors
selection
marker
For
eukaryotes
selection marker
For eukaryotes
Integration sequence
Integration sequence
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。
section 3
basic priciple and operation
of DNA recombination

basic principle
vector selection and
construction
obtaining of
purpose gene
recombinant DNA
transferring to cells
the connection of exogenous
gene and vector
the screening of
recombinant DNA
Isolation
selection
the expression
of recombinant DNA
connection
transformation
screening and
identification
DNA cloning
recombinant DNA
transferring to host cell
bacteria
cultured in solid
culture media
screening ofbacterial
monoclone containing
recombinant DNA
I. isolation——the isolation and
obtain of purpose gene
1. chemical synthesis
2. genomic or cDNA library
3. PCR
4. other methods
1.chemical synthesis
primary structure
possible DNA
sequence
prediction!
www.ensembl.org
www.ensembl.org
ENSEMBL
EXON 1 of HOXD13
genomic DNA
RE
genomic fragment
2. genomic DNA library
clone vector
recombinant DNA
bacteria
bacteria containing
recombinant DNA
genomic
DNA
RE sequnce
R cos
cos L
RE
RE
internal sequence
R cos
cutting
cos L
left arm
right arm
~20 Kb DNA
reconbination
cos L
~20 Kb exogenous DNA
R cos
ligase
phage package
infect to E.coli
by recombinant
phage
genomic library
genomic library established by random cutting method
 cDNA library
AAAA
mRNA
reverse
transcription
cDNA
reverse
transcript
AAAA
ion
TTTT
replication
double strand
vector
recombinant DNA
bacteria
cDNA library
alkline
hydrolysis
TTTT
DNA
polymeraseⅠ
SI nuclease
3. PCR
ATG
TGA
ATG
TGA
F
R
II.rebuild of vector
 purpose：
① to obtain a ppurpose gene fragment
② to obtain protein encoded by the gene
different clone capacity and suitable host
vector
DNA insertion
host cell
plasmid
<10kb
bacteria, yeast
λ-phage
~20kb
bacteria
cosmid
~50 kb
bacteria
BAC
~400kb
bacteria
YAC
~3 Mb
yeast
BamHI ATG
SalI
SmaI
XbaI
TAG
EcoRI
BamHI
EcoRI
10* H buffer
1 μl
1 μl
1 μl
Vector /
gene fragment
ddH2O
total volume
up to 1 μg
fill up to 20 μl
20 μl
purification of DNA
BamHI
Vector
EcoRI
III. connection of vector and gene fragment
1. connection of sticky ends
（1）single sticky ends
（2）different sticky ends
（3）sticky ends generated by other methods
single sticky ends
GGATCC
CCTAGG
G
CCTAG
GATCC
G
Bam HⅠcutting purpose gene
G
CCTAG
G
CCTAG
GGATCC
CCTAGG
GGATCC
CCTAGG
self connection
of vector
Bam HⅠ
+
GGATCC
CCTAGG
GGATCC
CCTAGG
Bam HⅠcutting vector
GATCC
G
GATCC
G
T4 DNA ligase
15ºC
recombinant
DNA
GGATCC
CCTAGG
GGATCC
CCTAGG
self connection of
purpose gene
different sticky ends
Eco RⅠ
GAATTC
CTTAAG
GAATTC
CTTAAG
Bg lⅡ
AGATCT
TCTAGA
AGATCT
TCTAGA
EcoRⅠ+ Bg lⅡ
AATTC
G
A
TCTAG
Eco RⅠ+ Bg lⅡ
AATTC
G
GATCT
A
T4 DNA ligase
15ºC
GAATTC
CTTAAG
AGATCT
TCTAGA
recombinant
DNA
+
AATTC
G
A
TCTAG
sticky ends of gene fragment generated by PCR
artificial linker
F
exogenous DNA
EcoR I
R
2. connection of flat ends
Suitable for：
•Flat ends generated by the cutting of RE
• flat ends generated by the sticky end filling
DNA polymerase I
G
CCTAG
GGATC
CCTAG
Purpose DNA
vector
RE
RE
T4 DNA ligase
15ºC
Recombinant
DNA
Self connection
Of vector
Self connection
Of fragment
3. Connection of sticky and flat ends
SmaⅠ
Bg lⅡ
CCCGGG
GAATTC
GGGCCC
CTTAAG
AGATCT
TCTAGA
CCCGGG
GAATTC
GGGCCC
CTTAAG
AGATCT
TCTAGA
SmaⅠ+ Bg lⅡ
GGG
AATTC
CCCG
A
TCTAG
smaⅠ+ Bg lⅡ
CCC
AATTC
G
GGG
GATCT
A
T4 DNA ligase
15ºC
GAATTC
CCCGGG
CTTAAG
GGGCCC
AGATCT
TCTAGA
recombinant
DNA
+
GGG
AATTC
G
CCC
A
TCTAG
Ⅳ. The transferring of recombinant DNA
The features of host cells：
Safe host
Lack of RE and recombinant E
Competent cell
The methods of transferring：
Transformation
Transfection
infection
Ⅴ. The identification of recombinant DNA
1. By the genetic marker on the vector
(1) Antibiotics marker
(2) Specific sequence
RE digestion
PCR
nucleic hybridization
DNA sequencing
summary
DNA recombination technology ：
isolation
Isolation of purpose gene
selection
Selection of vector
connection
Connection of fragment and vector
transformation
Recombinant DNA transferring to host cell
Identification
Screening and identification of
recombinant DNA
Ⅵ. The expression of recombinant DNA
The establishment of expression system：
 the construction of expression vector
 the establishment of host cell system
 the isolation and purification of expression product
An expression vector is similar to
cloning vectors, but with a major
difference: the expression vector must
contain a promoter so that proteins can
be expressed.
Expression vector
Section 4
Application of Recombinant
DNA Technology in Medicine
biological pharmacy
 First gene engineering product –artificial synthesized
insulin from Eli Lilly company in US.
 The protein product or polypeptide product by using
gene engineering has become the most important
industry nowadays.
recombinant DNA product
product
function
Tissue plasminogen activator
anticoagulation
Blood coagulation factor VIII
coagulation
granule-macrophage colony
stimulating factor
Leukocyte growth stimulation
Growth factor
Cell growth and differenciation
Growth hormone
Nanism treatment
insulin
DM reatment
Interferin ( 1b, 2a,  2b, )
Anti-tumor or anti-virus
Monoclonal antibody
Specific detection of diagosis
experiment, tumor target therapy
Hepatitis B immunology
Prevention of hepatitis B
Other applications of recombinant
DNA technology in medicine
the explore and indentify of pathogenic gene
 transgenic engineering and gene targeting
 accelerate the HGP
 gene diagnosis and gene therapy