dna technology. heredity. dna is very stable, and genetic information can be faithfully passed down...
TRANSCRIPT
DNA Technology
Heredity. DNA is very stable, and genetic
information can be faithfully passed down to
new generations.
Evolution. However, genetic information can
be exchanged laterally across cells or
species. Exchanging genetic information is
the reason of biodiversity.
Gene manipulation needs gene sequence and special proteins.
sequence “We, the Heads of States of the United States of America, the United Kingdom, Japan, France, Ger-many and China, are proud to announce that scientists from these six countries have completed the essential sequences of 3 billion base pairs of DNA of the human genome…”
- Joint Proclamation on HGP 14 April, 2003
Section 1
Genetic engineering
Cloning is the process of producing populations of
genetically-identical individuals asexually.
Cloning in biotechnology refers to processes used
to create copies of DNA fragments (molecular
cloning), cells (cell cloning), or organisms.
Molecular cloning refers to the procedure of
isolating a defined DNA sequence and obtaining
multiple copies of it in vitro.
Recombinant DNA is introduced through the
addition of a relevant DNA fragment into another
DNA sequence.
Genetic engineering, also known as recombinant DNA technology,
gene cloning, molecular cloning, genetic modification/manipulation
(GM) that apply to the direct manipulation of an organism’s genes.
Genetic engineering is different from traditional breeding, where the
organism's genes are manipulated indirectly;
Genetic engineering: is the technique manipulated on a molecular le
vel. The target gene and vector are ligated, introduced into the reci
pient cells which multiply the recombinant DNA molecule and expre
ss the protein products coded by the target gene.
Genetic engineering techniques have found some successes in num
erous applications, such as medicine, forensic science, environment
al science.
Genetic engineering
Big events
1953 Watson, Crick proposed DNAdouble helix
1970 Smith, Boyer found restriction endonuclease
1972 P.Berg , the first recombinant DNA
1973 S.N.Cohen Construction of the first biologically
functional bacterial plasmid in vitro
1975 Gilbert and DNA sequencing
1978 Human insulin produced by gene engineering
1985 Genetic modificated animals
1986 Mullis discovered PCR
1989-2002 Human genome sequencing
Paul Berg
Hebert Boyer
Stanley Cohen
I
II
III
IV
V
Procedures of genetic engineering
1. Basic tools used for genetic engineering
Toolenzymes
restriction endonucleases
DNA ligase
other enzymes
vectors
Host cells
Methods for recombinants screening
(二)重组 DNA 技术中常用的工具酶
Enzymes Functions
Restriction endonuclease
Recongizes and cut a specific DNA sequence
DNA ligase ligation of two DNA molecules
DNA polymeraseⅠ Synthesis of dsDNA
Reverse transcriptase
Synthesis of cDNA from RNA template
Polynucleotide kinase
32P labeling of DNA or RNA
Terminal transferase
Addition of poly A tail
Alkaline phosphatase
Dephosphate 5 end of DNA or RNA
Common tool enzymes
(1) Restriction endonucleases
Restriction endonucleases: the enzymes syn
thesized by bacteria can recognize and cleav
age specific nucleotide sequences
Hin dⅢ
Genus Species Strain Order
The third enzyme found in Haemophilus influenzae d strain
Nomination
restriction/modification systems
“ restriction system” of bacteria can destroy any "no
n-self" DNA
“modification system” can recognize “self” DNA an
d protect it by methylating it (methylase).
Restriction enzyme destroys unprotected (non-self)
DNA.
Category of restriction endonucleases
Category ATP ComponentsRecognition sequence Cleavage site
Methylase activity
I necessory Three kinds of subunits
asymmetrical differs, and is some distance (at least 1000 bp) away, from their recognition site
Y
II unnecessory Single poly-peptide
Palindromic,4-8 nucleotides
Cut at the same site N
III necessory Two kinds of subunits
Cut about 20-30 base pairs after the recognition site
Yasymmetrical
Recognition site of restriction endonucleases
Palindrome: is reversed repeat sequence in dsDNA,
namely the recognition site reads the same on the re
verse strand as it does on the forward strand.
5’ GGATCC 3’3’ CCTAGG 5’
5’ GAATTC 3’3’ CTTAAG 5’
5’ CCCGGG 3’3’ GGGCCC 5’
BamH I
EcoR I
Sma I
Recognition siteCut site
The ends produced by restriction endonucleases digestion
Bam HⅠ
GTCCAG
GCCTAG
GATCC G++
GGATCCCCTAGG
HindⅡGTCGACCAGCTG
GACCTG++
Blunt ends Sticky ends
reaction : break phosphate diester bond at a special site
Frequently used restriction endonucleases
(2) DNA ligase
DNA ligase: a kind of enzyme that can link together t
wo DNA strands that have double-strand break (a bre
ak in both complementary strands of DNA).
The mechanism of DNA ligase is to form two coval
ent phosphodiester bonds between 3’ hydroxyl ends o
f one DNA fragment with the 5’ phosphate end of anot
her. ATP is required for the ligase reaction.
T4 DNA ligase, ATP T4 DNA ligase, ATP
DNA polymerase is an enzyme that catalyzes the polyme
rization of deoxyribonucleotides into a DNA strand.
(3) DNA polymerase
DNA polymerase I of E.coli
5’-3’polymerase
5’-3’exonuclease
3’-5’exonuclease Klenow enzyme
Cleave site of protease Klenow enzyme
36 kDa 5’ 3’exonuclease 76 kDa 5’ 3’ polymerase
3’ 5’ exonuclease
protease
N C
T4 DNA polymerase
T7 DNA polymerase
Taq DNA polymerase thermal stable
5’-3’polymerase
3’-5’exonuclease
Reverse transcriptase
DNA polymerase
dTTP 3'
3' A T G C A A T T G C 5 '
| | | |
5 T A C G
ppi
T
AAAA 3’5’ m7Gppp
Oligo(dT), AMV RT, dNTPs
RNase H, DNA polymerase I
5’ m7Gppp AAAA 3’TTTT 5’
5’ m7Gppp AAAA 3’TTTT 5’
3’
3’
AAAA 3’TTTT 5’3’
5’
T4 DNA polymerase, dNTPs, DNA ligase
cDNA
cDNA synthesis catalyzed by reverse transcriptase
(4) vectors
Vector: a small DNA vehicle into which a foreign DNA fragment can be inserted, and which can be transferred into the recipient cells to replicate multiple copies or to express protein product.
Basic characteristics of vectors
① At least one origin of replication——replication
② Multiple cloning sites (MCS) ——insertion of target gene
③ At least one selectable marker gene—— recombinant screening
④ As small as possible
⑤ Be safe.
⑥ Expression vectors must possess corresponding elements (promoter,
enhancer)
Molecules origin:
plasmid
phage
cosmid
virus
Bacterial artificial ch
romosome, BAC
Yeast artificial chro
mosome, YAC
Classification of vectors
Application area:
Vectors in Prokaryote
Vectors in Eukaryote
Functions :
Cloning vectors
Expression vectors
Shuttle vectors
A cloning vector is a small piece of DNA into which a foreign DNA
fragment can be inserted, and which can be transferred into the
host cells to replicate multiple copies of it.
The main purpose of these vehicles is the replication of a particular
gene inside a convenient host organism.
An expression vector is a DNA vehicle that is used to introduce a
specific gene into a target cell and to express the protein encoded
by the gene.
The main purpose of these vehicles is the controlled expression of
a particular gene inside a convenient host organism.
A shuttle vector is a vecotor (usually a plasmid) constructed so th
at it can propagate in two different host species.
The main advantage of these vectors is they can be manipulated in
E. coli and then used in a system which is more difficult or slower t
o use (e.g. yeast, other bacteria).
A plasmid is an extra-chr
omosomal circular double-
strand DNA molecule sepa
rated from the chromosom
al DNA of bacteria which is
capable of replicating inde
pendently of the chromoso
mal DNA ( 1 kb - 500 kb
) .
plasmid
Characteristics of plasmids : Origin of replication (ori)
stringent plasmid : low copy number, several/cell
relaxed plasmid : high copy number, 10-200/cell
Multiple cloning sites ( MCS ) Selectable marker genes
(antibiotic resistance genes, lacZ’ gene)
As small as possible for inserts of about 1-10 k
bA multiple cloning site (MCS) is a short DNA segment
which contains many (up to ~20) unique restriction sites
for insertion of the target DNA sequence.
ApR: Ampicillin resistance gene
rep (ori): replication origin
lacZ: N-terminal -galactosidase
MCS: multiple cloning sites
MCS (pUC18 )
relaxed ori, 500-700 copies /cell antibiotic resistance gene and lacZ’gene MCS 2686bp
pUC plasmid
Bacteriophages
Bacteriophages are v
iruses that infect bacte
ria.
Typically, bacteriopha
ges are much smaller
than the bacteria, con
sist of an outer protein
capsid enclosing gene
tic material.
Lysogenic cycleLytic cycle
induction
bacteriophage DNA
E.coli DNA
new DNA
The infection cycle of bacteriophage λ
Genome of λ bacteriophages
double-stranded linear DNA
5’ TCCAGCGGCGGGG 3’
3’ CCCGCCGCTGGA 5’ COS
COS
COS site: The extreme
ends of the λ DNA are stic
ky sites with 12-nucleotide
s 5’ overhangs. When λ ph
age infects the cell, The lin
ear dsDNA immediately cir
cularises using the cos sit
es.COS site
Insertion λ phage vectors (λgt10 and λgt11)
replacement λ phage vectors (EMBL3 and EMBL4)
Up to 25 kb of
foreign DNA can
be cloned into
these vectors
Cosmid
Cosmid is a type of hybrid pla
smid that contains cos sequen
ces from the λphage.
Cosmids are able to clone u
p to 45 kb of DNA.
Cosmids can replicate as pl
asmids if they have a suitable
origin of replication, and they c
an also be packaged in phage
capsids.
Bacterial artificial chromosome (BAC)
Bacterial artificial chromosome (BAC) is a
DNA construct, based on a functional fertility
plasmid (contain partition genes), used for cl
oning 150-350 kb DNA.
BACs are often used to sequence the gen
ome of organisms in genome projects.
Yeast artificial chromosome (YAC)
Yeast artificial chromosome (YAC) is a vect
or used to clone large DNA fragments (up to 3
000 kb). It is an artificially constructed chrom
osome and contains the telomeric, centromeri
c, and ori sequences needed for replication a
nd preservation in yeast cells.
Vectors Size DNA insert Uses
Plasmid 4.6 kb 0.1-10kb Cloning, expression
λphage 50 kb 8-25 kb Library building
Cosmid 5- 7 kb 35 – 45 kb Library building
Yeast aritificial chormosome
8 kb 100-1000 kb Library building
Virus varied varied In eukaryotes
Common cloning vectors
Expression vectors
PET expression vectors
T7 promoter lacO RBS MCS T7 terminator
expression Target gene
(5) Host cells
Prokaryotic cells
Simple Eukaryotic cells
Plant and animal cells
Common host cells——E.coli
advantages : 1. genetic simplicity (sequenced, 4600 kb, 4000 genes)
2. fast growth rate (1 generation / 20 min)
3. safety
4. single cell
Disadvantages :
1. no protein folding system
2. quantities of endogenous proteases
3. no protein precessing system
Common host cells——fungal cells
advantages :
1. eukaryote with simple structure
2. protein processing system after translation and
secretion system
3. safety
4. easy culture
Common host cells——plant cells
advantages : totipotency
application : transgenic plant
Animal cells
advantages :
1. mRNA splicing system
2. protein processing system
3. easy transfection
4. protein secretion
disadvantages : stringent conditions of cultivation
2. Procedure of genetic engineering
(1)seperation——
to obtain target gene or interested DNA fragment
(2)digestion——
cut to obtain target gene with proper ends and cut the vector with restriction enzymes
(3)ligation——
to ligate the target gene and the vector to form recombinant DNA
(4)transformation——
introduce the recombinant DNA into host cells
(5)screening——
to select transformant colonies that contain the vector with target gene
(6)expression——
to express target gene under the control of expression vector
Cloning vector
plasmid phage virus
Target gene
Restriction endonuclease
Linear vector Linear gene fragment
DNAligation
Recombinant DNA
transformation transfection packing in vitro
Host cells
phenotype electrophoresis hybridyzation immuno-reaction
Expression target gene
PCR cDNA synthesis gene library
Procedure of genetic engineering
Cloning vector
Restriction endonuclease
DNA ligase
Recombinant vector
Transformation or transduction
E.coli
Amplification of host cells
Recombinant vector replicates in host cells to form many copies
Basic steps of gene engineering
Obtaining the target gene
• Chemical synthesis
• Gene library
• cDNA library
• Polymerase chain reaction (PCR)
(1) Obtaining the target gene
(2) Restriction enzyme digestion
Construction of gene library
(3) Ligation
(4) Introduce recombinant DNA into host cells
Transformation : the process of bacteria taking up re
combinant DNA constructed from plasmid.
Transduction : the process of bacteriophage injectin
g the foreign DNA into their bacteria.
Transfection : the process of viruses injecting the for
eign DNA into Eukaryotic cells.
Transfection: the process of viruses injecting the forei
gn DNA into animal cells.
In addition, there are still other methods, such as particl
e bombardment and microinjection.
CaCl2 transformation
Host bacteria
50-100mmol/L
competent bacteria Bacteria carrying recombinant DNA
CaCl2
Chilling cells in the presence of CaCl2 prepares the cell membrane
to become permeable to plasmid DNA. Cells are incubated on ice wi
th the DNA and then briefly heat shocked (eg 42 ℃ for 30–120 sec),
which causes the DNA to enter the cell.
This method works very well for circular plasmid DNAs.
Competent cells refer to cells in the state of being able to
take up exogenous DNA from the environment.
Genomic DNA
plasmid DNA
Heat shork (42℃)
Electroporation is a significant increase in the e
lectrical conductivity and permeability of the cell
plasma membrane caused by an externally appli
ed electrical field.
In molecular biology, the process of electropor
ation is often used for the transformation of bact
eria, yeast, and plant protoplasts.
Electroporation
cuvettes for electroporation
electroporator
DNA
cell transformedcell
Transduction
Gene recombination
transduction
In vitro packaging
bacteriophage
Particle bombardment microinjection
(5) Screening of transformed cells
Transformant (b, c, d, e): host cells carrying exogenous DNA.
Recombinant (c, d): transformant carrying recombinant DNA.
Positive recombinant (c): recombinant carrying exogenous
target gene.
a b c
d e
Screening by genetic phenotype
—— antibiotic resistance
TetInserted fragment
Amp plate
Tet plate
Amp
antibiotics
stocking solution working solution
concentration Store Tstringent plasmid
relaxed plasmid
Ampicillin
( Ap/Amp )50 mg/ml (H2O) -20°C 20 μg/ml 60 μg/ml
Chloromycin
( Cm/Cmp )34 mg/ml (ethanol) -20°C 25 μg/ml 170 μg/ml
Kanamycin
( Kn/Kan )10 mg/ml (H2O) -20°C 10 μg/ml 50 μg/ml
Streptomycin
( Sm/Str )10 mg/ml (H2O) -20°C 10 μg/ml 50 μg/ml
Tetracyclin*
( Tc/Tet )5 mg/ml (ethanol) -20°C 10 μg/ml 50 μg/ml
Antibiotics
—— Blue/white screening
Screening by genetic phenotype
E. coli β-galactosidase gene is most widely used as a marker gene, w
hose integrity can easily be detected by the ability of the enzyme it enco
des to hydrolyze the soluble, colourless substrate X-gal (5 bromo-4-chlor
o-3-indolyl-beta-d-galactoside) into an insoluble, blue product (5,5'-dibro
mo-4,4'-dichloro indigo).
This enzyme can be split in two peptides, LacZ’(146 amino acids at N
terminal) and LacZ’’, none of which is active by itself but both spontaneo
usly reassemble into a functional enzyme. This characteristic is used in
many cloning vectors to achieve α-complementation in specific labor
atory strains of E. coli, where the small LacZ’ peptide is encoded by the
plasmid while the large LacZ’’ is encoded by the bacterial chromosome.
When target DNA fragment is inserted in the vector and production of La
cZ’ is disrupted, the cells exhibit no β-galactosidase activity: this allows t
he transformant colonies in the selective agar plates white, while blue in
the case of a vector with no inserted DNA.
Screening by structure feature of recombinants
Agarose gel electrophoresis
DNA Marker
plasmid
Recombinant plasmid recombinant plasmid digested by RE
PCR product of recombinant plasmid
(6) Expression of the cloned gene
recombinant
mRNA of target gene
Expressed protein
E.coli
Function analysis
Structure analysis
Construction of drugs screening
model
Clinical medicine
Gene therapy
Expression target protein in E.coli
SDS-PAGE (a) Western blot (b)
M1 1 M2 2 3 M1 1 M2 2 3
3. Application of genetic engineering
gene engineered medicine
gene therapy
gene chip
transgenic plant
transgenic animal
environment and energy resources
• Human insulin (1982 Ely LiLi, USA)
• Human interferon (hIFN)
• Hematopoietin (EPO)
• Human interleukin (hIL)
• Streptokinase (SK)
• Urokinase ( UK )• Tissue plasminogen activator ( t-P
A )• Gene engineered antibody
• Gene engineered vaccine
Gene engineered medicine