Genetic Engineering

--- started in 1973

What is genetic engineering?
A process in which an isolated and
well characterized gene is
transfered, integrated and
expressed in a host cell
What is DNA recombination?
It refers to the process in which
two independent DNA molecules
are covalently ligated to form a
new DNA moleculer.
 Insulin + vector
gene
ligation
transformation

E.coli
Screening,
amplifying

expression

rhInsulin lysing 、 purification

 Insulin

Insulin is secreted into milk

Expression of Insulin is
restricted to mammary
tissue

Pure insulin product

Ⅰ The tool enzymes of GE
1.Restriction endonuclease
2. DNA ligase
3. DNA pol-Ⅰ
4.Reverse Transcriptase
5.Klenow fragment
6.Alkaline phosphatase
7.Terminal deoxynucleotidyl
thansferase,TdT
8.Taq
1.Restriction Enzymes
Enzymes that recognize specific DNA
sequences and cleave them to
produce DNA fragments of varying
length.

ATACCACCAGGGTTACAGGATAGGAGTCAGGATCCAGAGGACCTAGGAT
TATGGTGGTCCCAATGTCC TA TCCTCAGTCCTAGGTC TC CTGGATCCTA
1.Restriction Enzymes
One may use restriction enzymes as a type
of "molecular scissors" to excise a desired
DNA fragment and transfer it into a cloning
vector.
The ability to specifically cleave DNA
sequences at known sites underlies many of
the advances made over the last several
decades in molecular biology.
Recognition sequences
Recognize 4-8 bp palindromic sequences. Most
commonly used enzymes recognize 6 bp which
occurs at a rate of 46=4096 bp. (44=256 bp;
48=65536 bp)

e.g. EcoRI site: 5’ GAATTC 3’

3’ CTTAAG 5’
Restriction enzymes
1. Highly specific
2. Commercially available
3. Require Mg2+ for enzymatic activity
4. Compatible ends from different enzymes,
DNAase and RNAase cut up genetic
material at random.
Restriction enzymes cut only at certain
sequences of bases, called restriction sites.
DNA double helixes are cut at the
axis of symmetry:
 Two kinds of ends :
Some make “blunt” ends: ATTC GGATC
TAAG CCTAG

Some make “sticky” ends: ATTCGG

ATC
TAA GCCTAG

These pieces are restriction fragments.

Commonly used Recognition sequences
restrict enzymes and cleaving sites
EcoR Ⅰ G↓AATTC
HindⅡ GTPy↓PuAC
Hind Ⅲ A↓AGCTT
BsuR I GG↓CC
Pst Ⅰ CTGCA↓G
Sma Ⅰ CCC↓GGG
Xba Ⅰ T↓CTAGA
Xho Ⅰ C↓TCGAG
BamHⅠ G↓GATCC
Not Ⅰ GC↓GGCCGC
2. DNA ligase
A enzyme that joins two double-
stranded DNAs end to end.
In nick-sealing reaction
1). Ligation of sticky ends

5′ 3′ 5′ 3′
*********G —OH P— A A T T C*********
*********C T T A A — P OH— G*********
3′ 5′ 3′ 5′

5′ 3′
DNA ligase **********GAATTC**********
**********CTTAAG**********
EcoR I 5′
3′
2). Ligation of blund ends

5′ 3′ 5′ 3′
************TC —OH P — CT*************
************AG — P OH— GA*************
3′ 5′ 3′ 5′

5′ 3′
DNA ligase *************AGCT**************
*************TCGA**************
Alu I 3′ 5′
3. DNA pol-Ⅰ
One of the three different DNA-
synthesizing enzyme in E.coli; used
primarily in DNA repair.
Synthesis of double-stranded cDNA
;
4.Reverse Transcriptase
RNA-dependent DNA polymerase
Catalyzes reverse transcription.
Synthesis of cDNA from mRNA.
5.Klenow fragment
A fragment of DNA polymeraseⅠ,
created by cleaving with a protease,
that lacks
5’ 3’ exonuclease activity of the
parent
enzyme.
6.Alkaline phosphatase
Remove the 5’-phosphates.
Prevent the vector from self-ligation.
7.Terminal deoxynucleotidyl
thansferase ,TdT

A enzyme that adds

deoxyribonucleotides , one at a
time, to
the 3’-end of a DNA.
Homopolymer tail
1. ) Single stranded-DNA
or 3′-sticky end

5′ 3′ Mg2+ 5′ 3′
OH (A 、 G 、 C 、 T)n
dNTP ppi
n

Mg2+
OH (A 、 G 、 C 、 T)n
5′ 3′ dNTP 5′ 3′
nppi
2). Blund end

5′ 3′ Co2+ 5′ 3′
OH (A 、 G 、 C 、 T)n
dNTP nppi

Co2+
OH (A 、 G 、 C 、 T)n
5′ 3′ dNTP ppi 5′ 3′
n
How to get the gene from species A to
species B?

A vector is used to carry the gene into the

host nucleus.
Ⅱ Vector
A piece of DNA (a plasmid, a phage
DNA or virus) that serves as a
carrier in genetic engineering.
Cloning vector: amplifying the
cloned gene.
Expression vector: allowing
expression of the cloned gene.
What are vectors commonly used
in GE?
1.Plasmid (of bacteria)
2.Phages
3. cosmids
4. Viruses
5.yeast artificial chromosomes
 1.What is a plasmid?

A plasmid is a small, extra-chromosomal,

circular molecule of DNA that replicates
independently of the host DNA.
Natural role of a plasmid in
bacteria?
Plasmids usually contain one or two
gene that confer a selective
advantage on the bacterium e.g.
antibiotic resistance gene.
Every plasmid possess a replicator
gene can duplicated independently
from the chromosomal DNA.
The plasmids typically have three
important elements:
A cloning site (a place to insert
foreign DNAs)
An origin of replication
A selectable marker gene (e.g.
resistance to ampicillin)
 pBR322 Plasmids as Cloning Vectors
ORI ： High copy
number (1000-3000
copies per cell)

Ampr, Tetr ： Ampr

Two antibiotic resistance
markers for positive selection Tetr

Pst I, BamH I ：
cloning sites for inserting ORI
foreign DNAs
Plasmids as Cloning Vectors
Small, only 4361 BP,
Stable in E. coli,
High copy number (1000-3000
copies per cell)
Easily isolated in the
supercoiled form
Foreign DNA can be inserted
in good amount
Plasmid pBR322 Restriction sites are known
Single cleavage sites for
several restriction enzymes
Two antibiotic resistance
markers
Transformation easy
 pcDNA3.0 as Expression Vectors

Pcmv ：
CMV enhancer/Promoter
Driving
target gene
expression
Hosts for cloning vectors
Ideal hosts: rapid growth, capable of growth in cheap
culture medium, not harmful or pathogenic,
transformable by DNA, stable.
Prokaryotic hosts: E. coli, Bacillus subtilis.
Eukaryotic hosts: yeast
DNA virus SV40, a virus causing tumors in primates,
can be used as a cloning vector into human culture
lines.
Retroviruses, vaccinia virus are useful too.
Baculovirus, an insect DNA virus can be used to
transfer DNA to insect cell lines
Ⅲ The process of GE
1.Separating:
2.Cleaving:
3.ligating:
4.Transferming:
5.Screening:
6.Expressing:
1.Separating the target gene
1)chemical synthesis
2)PCR and RT-PCR
3)gene library: Genomic library and

cDNA library
Genomic library:
A collection of clones made from a
set of randomly generated
overlapping DNA fragments
representing the entire genome of
an organism.
cDNA library:
A collection of cDNA clones that
were generated in vitro from the
total mRNA sequences isolated
from an organism or a specific
tissue or cell type or population of
an organism.
2.Cleaving the vector
Using restriction enzyme
3.Ligation of the target gene and vector
1)Sticky end ligation

Target gene
Restriction enzyme
cutting

DNA ligase

same enzyme
cutting sites
Recombinant plasmid
2)blunt end ligation

Restriction enzyme
cutting
S1
S1
ligase

No same enzyme
cutting sites
Recombinant
plasmid
3)Linker ligation + + linker

ligase

Same restriction
enzyme cutting

ligase

No same enzyme
cutting sites ！
Recombinant plasmid
4)Homopolymeric tail ligation

Target gene
Cutting
TdT TdT
+dGTP +dCTP

ligase

No same enzyme
Recombinant plasmid cutting sites
4.Transfering the recombinant
plasmid into host
E.coli
0 ～ 4℃ CaCl2
Competent cell
recombinant
plasmid transformation
E.coli containing
recombinant plasmid
How do I know which bacteria will have the
plasmid?
Add specific antibiotics to the agar
culture
All those bacteria which do not
have the plasmid will die
Only those with the recombinant
plasmids can grow and multiply
5 、 screening the recombinant
DNA
1) Insertion disruptting: insert target gene in Ampr

Ampr
disrupt the Amp Terr
resistance gene
eg ：

1 2 3 1 2 3

4 5 6 4 5 6

Tet Amp
3 and 5 are positive clone
2) ： blue-white screening

Apmr

MCS
Insert target gene in
the Laz gene
β -galactosidase LacZ
 2) ： blue-white screening Lac-Z gene
β -galactosidase
X-gal Blue products

White clones
are positive!

α-complementation
6.Expression of target protein.
 Cut insulin gene from
Cut plasmid
human cell
(by adding
(by adding restriction
restriction
enzyme)
enzyme)

adding ligase enzyme

Insulin gene has been inserted into the plasmid

 Place the recombinant plasmids back
into the bacteria

Leave the bacteria to grow and multiply

Ⅳ.Application of GE:
Medical

GM Food
Industrial production
Environmental protection
Medical
Production of pharmaceuticals for
treatment of diseases e.g. human
insulin, interferons

Production of pharmaceuticals for

disease prevention e.g. vaccine
(hepatitis B vaccine)
Medical
Gene therapy:
Artificially replace the disease-
causing gene with a normal allele.
The normal allele can be carried by
a virus vector to the target tissues.
e.g. treatment of cystic fibrosis
Medical
Clonal propagation:
a source of tissue or organ for
transplantation
avoid all problems of
immunoincompatibility.
Agricultural
Transgenic plants and farm animals
pest-resistant (reduce use of pesticides),
increase yield

Increase storage time

e.g. green tomato
tomato with beef genes
Industrial:
Use of GM microorganisms to
make stone-wash jeans

Use of GM microorganisms to
produce enzymes e.g. detergents
Environmental protection
GM E. coli possesses gene to break down
cellulose, speeding up recycling of the most
abundant biomass on earth

GM microorganisms with enhanced

ability to break down
environmental pollutants
Dispute in the development of GM
Dangerous pathogens formed in the
course of rDNA
New tools for militarists and terrorists
Triggering of catastrophic ecological
imbalance
Moral problems in the use of GM
techniques in man e.g. germ cell
gene therapy
Unknown effect of GM food on men
Thank you very much! Bye-bye!
( 二 ) 反转录法： 加热变性

TTnT 5′
5′ AAnA 3′ 加入特异性引物
mRNA
反转录酶
3′ cDNA
TTnT 5′
AAnA 3′
5′ mRNA DNA 聚合酶
核糖核酸酶 H
3′ cDNA
TTnT 5′
DNA poly I
重复上述步骤
核酸酶 S1 扩增出大量的产物
聚合酶链式反应 (Polymerase
双链 cDNA Chain Reaction ， PCR)
• Genes dictate proteins that are made.
• Proteins made dictate properties of cell or organisms.
• Each human has same genes, but genes can vary in
sequence between individuals. (Alleles)

Gene for brown hair

Genotype AGACTACACTG
G
Change in brown
hair gene (mutation/genetic differences)

Phenotype
Figure 12.17 Sickled and
Normal Red Blood Cells
What is genetic engineering?

The transfer of genes (segment of DNA)

from one species to another.
This is impossible in naturally breeding.
The process of altering the genetic
material of a cell or organism so as to
change its ability to function or to produce
new gene products