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