Genetic Engineering(1)using

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 gene

+ ligation

vector 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)

5’ GAATTC 3’ e.g. EcoRI site: 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 restrict enzymes EcoR Ⅰ HindⅡ Hind Ⅲ BsuR I Pst Ⅰ Sma Ⅰ Xba Ⅰ Xho Ⅰ BamHⅠ Not Ⅰ

Recognition sequences and cleaving sites G↓AATTC GTPy↓PuAC A↓AGCTT GG↓CC CTGCA↓G CCC↓GGG T↓CTAGA C↓TCGAG G↓GATCC GC↓GGCCGC

2. DNA ligase A enzyme that joins two doublestranded DNAs end to end. In nick-sealing reaction

1). Ligation of sticky ends 5′

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

DNA ligase EcoR I

5′

3′

P— A A T T C********* OH— G*********

3′

5′

5′ **********GAATTC********** **********CTTAAG**********

3′

3′

5′

2). Ligation of blund ends 5′ 3′ ************TC —OH ************AG — P 3′ 5′

DNA

ligase

Alu I

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

5′ 3′ *************AGCT************** *************TCGA************** 3′

5′

3. DNA pol-Ⅰ One of the three different DNAsynthesizing 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′ OH

Mg2+

5′

3′ (A 、 G 、 C 、 T)n

ppi

dNTP

OH 3′ dNTP

5′ n

Mg2+ nppi

5′

(A 、 G 、 C 、 T)n

3′

2). Blund end 3′ OH

5′

5′

Co2+ dNTP

3′ (A 、 G 、 C 、 T)n

ppi

n

Co2+ 5′

OH

3′

dNTP

n

ppi

5′

(A 、 G 、 C 、 T)n 3′

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 foreign DNAs

ORI

Plasmids as Cloning Vectors

Plasmid pBR322

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 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

Recombinant plasmid

No same enzyme cutting sites ！

4)Homopolymeric tail ligation Target gene

Cutting

TdT

TdT +dCTP

+dGTP

ligase

Recombinant plasmid

No same enzyme cutting sites

4.Transfering the recombinant plasmid into host E.coli 0 ～ 4℃ CaCl2

Competent cell recombinant transformation plasmid 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 resistance gene

Terr

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

Insert target gene in the Laz gene β -galactosidase

MCS

LacZ

2) ： blue-white screening

Lac-Z gene

β -galactosidase X-gal

White clones are positive! α-complementation

Blue products

6.Expression of target protein.

Cut plasmid

Cut insulin gene from human cell

(by adding restriction enzyme)

(by adding restriction 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 diseasecausing 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!

加热变性

( 二 ) 反转录法： 5′

TTnT 5′ AAnA 3′

mRNA 反转录酶 3′ cDNA TTnT 5′ AAnA 3′ 5′ mRNA 核糖核酸酶 H 3′ cDNA TTnT 5′ DNA poly I 核酸酶 S1

双链 cDNA

加入特异性引物

DNA 聚合酶

重复上述步骤

扩增出大量的产物 聚合酶链式反应 (Polymerase 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