chapter 17 gene technology. protein rna dna transcription translation cctgagccaactattgatgaa...
TRANSCRIPT
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Chapter 17
Gene Technology
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Protein
RNA
DNA
transcription
translation
CCTGAGCCAACTATTGATGAA
PEPTIDE
CCUGAGCCAACUAUUGAUGAA
Central Dogma: DNA -> RNA -> Protein
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Genetic Recombination in Humans
There are three ways in which meiosis and fertilization ensure that a child has a different combination of genes from that of either parent:
1. Independent assortment of chromosomes during metaphase I
2. Crossing-over during prophase I3. Upon fertilization, recombination of chromosomes
from different individuals (via their gametes) occurs.
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Recombinant DNA technology or genetic engineering was developed in 1971-1973
Their core was gene cloning Lead to DNA sequencing techniques that
enabled the structures of individual genes to be
determined Lead to procedures for studying the regulation
of individual genes
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Genetic recombination - transfer of DNA from one organism
(donor) to another recipient. The transferred donor DNA may then be integrated into the recipient's nucleoid by various mechanisms (homologous, non-homologous).
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Types Of Recombination
Generalized or Homologous Recombination - Occurs during prophase of meiosis I and involves exchange between homologous strands of DNA
Site Specific Recombination - Short homologous sections of bacterial and phage DNA serve as a site for recombination and thus incorporation of phage DNA into bacterial chromosomes
Transposition - Not truly recombination between different genomes, but the movement of transposons within a genome
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Homologous recombination-
homologous DNA sequences having nearly the same
nucleotide sequences are exchanged by means of Rec
A proteins. This involves breakage and reunion of
paired DNA segments as seen in Natural mechanisms
of genetic recombination in bacteria include:
a. transformationb. transductionc. conjungation
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The Current Prokaryotic
Recombination Model
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内切酶
(recBCD)DNA 侵扰(recA)
分支迁移 (recA)
内切酶(recBCD)
DNA 连接酶
5´ 3´
5´3´5´
3´
5´3´
5´ 3´
5´ 3´
5´3´
5´3´
5´ 3´
5´ 3´
5´3´5´3´
5´3´
5´ 3´
3´ 3´
5´3´
5´3´
3´5´
5´ 3´
5´3´
5´3´
Holiday 中间体
5´ 3´
5´ 3´
5´3´
5´3´
目 录
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Holiday 中间体
5´ 3´
5´ 3´
5´3´5´3´
5´3´
5´
5´5´
3´
3´
3´
5´ 5´
5´5´
3´
3´
3´
3´
5´ 5´
5´5´
3´
3´
3´
3´
5´5´
5´5´
3´
3´
3´
3´
5´ 5´
5´5´
3´
3´
3´
3´
内切酶(ruvC)内切酶
(ruvC)
DNA连接酶
DNA连接酶
片段重组
体拼接重组
体
Patch recombinationsplice recombination
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Genetic Transfer & Recombination In Bacteria
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Three kinds of genetic exchanges between prokaryotes
Three kinds Conjugation
Mediated by plasmids Transformation
Mediated by free DNA Transduction
Mediated by phages
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Bacterial Conjugation
Bacterial Conjugation is genetic recombination in which there is a transfer of DNA from a living donor bacterium to a recipient bacterium. Often involves a sex pilus.
The 3 conjugative processes
I. F+ conjugation
II. Hfr conjugation
III. Resistance plasmid conjugation
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F+ Conjugation- Genetic recombination in which there
is a transfer of an F+ plasmid (coding only for a sex
pilus) but not chromosomal DNA from a male donor
bacterium to a female recipient bacterium. Involves
a sex (conjugation) pilus. Other plasmids present in
the cytoplasm of the bacterium, such as those coding
for antibiotic resistance, may also be transferred
during this process.
I. F+ Conjugation Process
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Conjugal transfer of plasmid
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The 4 stepped F+ Conjugation
1. The F+ male has an F+ plasmid coding for a sex pilus and can serve as a genetic donor
2. The sex pilus adheres to an F- female (recipient). One strand of the F+ plasmid breaks
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The 4 stepped F+ Conjugation (cont’d)
3. The sex pilus retracts and a bridge is created between the two bacteria. One strand of the F+ plasmid enters the recipient bacterium
4. Both bacteria make a complementary strand of the F+ plasmid and both are now F+ males capable of producing a sex pilus. There was no transfer of donor chromosomal DNA although other plasmids the donor bacterium carries may also be transferred during F+ conjugation.
http://www.cat.cc.md.us/courses/bio141/lecguide/unit4/genetics/recombination/conjugation/f.htm l
F
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Transformation
Genetic recombination in which a DNA fragment from a dead, degraded bacterium enters a competent recipient bacterium and it is exchanged for a piece of the recipient's DNA.
Involves 4 steps
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1. A donor bacterium dies and is degraded 2. A fragment of DNA from the dead donor bacterium binds to DNA binding proteins on the cell wall of a competent, living recipient bacterium
3. The Rec A protein promotes genetic exchange between a fragment of the donor's DNA and the recipient's DNA
4. Exchange is complete
The 4 steps in Transformation
http://www.cat.cc.md.us/courses/bio141/lecguide/unit4/genetics/recombination/transformation/transformation.html
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Transduction
Genetic recombination in which a DNA fragment is transferred from one bacterium to another by a bacteriophage
Structure of T4 bacteriophage Contraction of the tail sheath of T4
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What are Bacteriophages?
Bacteriophage (phage) are obligate intracellular parasites that multiply inside bacteria by making use of some or all of the host biosynthetic machinery (i.e., viruses that infect bacteria
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An infection
cycle
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Transduction
There are two types of transduction: generalized transduction: A DNA fragment is
transferred from one bacterium to another by a lytic bacteriophage that is now carrying donor bacterial DNA due to an error in maturation during the lytic life cycle.
specialized transduction: A DNA fragment is transferred from one bacterium to another by a temperate bacteriophage that is now carrying donor bacterial DNA due to an error in spontaneous induction during the lysogenic life cycle
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Site Specific Recombination
Short homologous sections of bacterial and
phage DNA serve as a site for recombination
and thus incorporation of phage DNA into
bacterial chromosomes
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Integration of Lambda DNA-overview.
att = attachment site
INT = integrase
O = center core of 15 bases = the same in phage & bacterial
B,P = different in size and sequence in bacterial & phagedsDNA
XIS = Excisionase
The control of INT & XIS activity determines it latency or not.
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Integration of Lambda DNA-Detail of crossover
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例 : 细菌的特异位点重组
沙门氏菌 H 片段倒位决定鞭毛相转变
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Transposition
-Movement of gene to a new site, on same or a different chromosome
Does not require extensive homology
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Transposable elements
Insertion sequence
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插入序列的复制性转
座
目 录
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Transposons: Mobile genetic elements that enable genes to move between non-homologous sites in DNA –
Transposable elements. ⋅Altered expression of genes in new environments
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Gene technology
A set of methods and techniques used to study biological processes on the molecular level
There have been considerable developments in this field during the past two decades
Eg: new and powerful ways for the isolation, analysis, and manipulation of nucleic acids
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What is gene cloning
(1) A fragment of DNA is inserted into a circular DNA molecule
called a vector, to produce a chimera or recombinant DNA
molecule
The basic steps in gene cloning experiment are as follows:
(2) The vector acts as a vehicle that transports the gene into a
host cell
(3) Within the host cell the vector multiplies, producing
numerous identical copies
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(4) When the host cell divides, copies of the recombinant DNA molecule are passed to progeny and further vector replication takes place
(5) After a large number of cell divisions, a colony, or clone, of identical host cells is produced
Each cell in the clone contains one or more copies of the recombinant DNA molecule
The gene carried by the recombinant molecule is now said to be cloned
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The basic steps in gene cloning
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Why gene cloning is so important
Gene isolation by cloning Cloning allows individual fragments of DNA to be
purified isolated long genes or those that have never been studied
before
This technique can provide a pure sample of individual gene, separated from all the other genes in the cell
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Cloning allows individual fragments of DNA to be purified
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Clone A large population of identical molecules, or cells that arise from a common ancestor.
Cloning
The basic concepts about gene cloning
The process that produces a large number of DNA or cell copies
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vector target DNA Recombination DNA
Transformation
bacteria Recombination DNA
amplification
Transfer the bacteria to
a solid culture plate
Screening the bacteria containing
the recombinant DNA
The process of gene cloning with the
plasmid as vector
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DNA recombinationThe process that two DNA molecules from different source join together by covalent bond to form a new DNA molecule is called DNA recombination. Recombinant DNA
DNA recombination technique By the application of some tool enzymes, the target gene and vector are ligated together, then introduced into the recipient cells which multiply and express the protein products coded by the target gene, that is, DNA recombination technique, DNA cloning or Gene cloning, Molecular cloning.
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Genetic engineering
All the work or methods used related to
the gene cloning and the target gene expressed
in host cells to produce the special protein or
polypeptide, or to change the character of an
organism, are called genetic engineering.
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The requirements for the gene cloning
(1) The target genes
(2) The vectors
(3) The tool enzymes
(4) The host cells
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Target DNA
• cDNA
• Genomic DNA
The cDNA are synthesized by reverse transcriptionase based on their mRNA templates of a cell line or tissue.
It represents whole DNA sequence of a genome
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Purification of DNA from living cells
Preparation of total cell DNA (RNA)
Preparation of plasmid DNA
Preparation of bacteriophage DNA
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Cloning vector——Cloning vectors are DNAs which can carry target genes, transfer them into the recipient cells.
Cloning vector classes
Plasmid DNAPhage DNAVirus DNA
Vectors
As for the expression vectors, they can make the proteins which are coded by the target gene expressed in the host cell
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Plasmids Basic features of plasmids
Small (less than 10kb), Circular, duplex molecules of DNA
Exist at low or high copies within the bacteria, but useful plasmid present in multiple copies
Replicate independently from the bacterial cell
Contain selectable markers, eg: the antibiotic resistance capability conferred to bacterium
Possess at least one DNA sequence that act as an origin of replication Multiple RE sites ( multiple cloning sites, MCS)
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plasmid ( 质粒 )
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Fig pBR322 Go to pBR322
b
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Fig pUC19 Go to pUC
Origin of replication
Multiple cloning site (MCS)
Lac Z β-galactosidase gene
Ampicillin resistance gene
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Bacteriophages
Bacteriophages, or phages are viruses that specifically infect bacteria
Simple in structure, merely of a DNA (or occasionally RNA) carrying genes, including several for replication of the phage, surrounded by a protective coat or capsid made up of protein
Basic features of bacteriophages
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The general pattern of infection
Attaches to the outside of the bacterium and injects its DNA chromosome into the cell
The phage DNA is replicated, usually by specific phage enzymes coded by genes on the phage chromosome
Other phage genes direct synthesis of the protein components of the capsid, new phage particles are assembled and released
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Lytic cycle:
With some phage types the entire infection cycle is completed very quickly, possibly in less than 20 min. This type of rapid infection is called lytic cycle.
Lysogenic infection:
Characterized by retention of the phage DNA molecule in the host bacterium, possibly for many thousands of cell divisions
The result of Infection
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Common used phages
Bacteriophage λ
A linear dsDNA approximately 49 Kb in lengthAfter infection it forms circular structures The phage DNA is inserted into the bacterial genomeThe first two classes of vector to be produced were λ i
nsertion (λgt phages) and λ replacement (EMBL phage
s)
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Bacteriophage M13
A circular ssDNA, and has been used for sequencing of a cloned target DNA fragment
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Fig pUC19 Go to pUC
Origin of replication
Multiple cloning site (MCS)
Lac Z β-galactosidase gene
Ampicillin resistance gene
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Other vectors
Cosmid ( 粘性质粒 )
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Bacterial artificial chromosome (BAC) and yeast chromosome
Viruse are used as vectors, eg: retro-virus, adeno-virus, adenoassociated virus, etc
Other vectors
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The tool enzymes
Nucleases—cut, shorten or degrade nucleic acid molecules
Ligases—join nucleic acid molecules together Polymerase—make copies of molecules Modifying enzymes —remove or add chemical gro
ups Topoisomerases —introduce or remove supercoils
from covalently closed-circular DNA
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Nucleases degrade DNA molecules by breaking the phosphodiester bonds
There are two different kinds of nucleases
Exonucleases remove nucleotides one at a time fr
om the end of a DNA molecule
Endonucleases are able to break internal phospho
diester bonds within a DNA molecule
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ligases
To repair single-stranded breaks(discontinuities) that arise in double-stranded DNA molecules during DNA replication
Join together two individual fragments of double-stranded DNA
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Polymerases Synthesize a new strand of DNA complementary
to an existing DNA or RNA template Four types of DNA polymerase are used routinely
in genetic engineering
DNA polymerase I: from E.coli. Synthesizes dsDNA by formation of
a 5’,3’-phosphodiester bond
Klenow fragment : removes the first 323 amino acids from DNA poly
merase I , Synthesizes DNA by formation of a 5’,3’-phosphodiester
bond
Reverse transcriptase: synthesizes DNA from RNA template
Taq DNA polymerase: used in the PCR, it is the DNA polymerase I f
rom bacterium Thermus aquaticus
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DNA modifying enzymes
Alkaline phosphatase from E.coli, calf intestinal tissue or arctic shrimp removes the phosphate group present at the 5’term
inus of a DNA molecule
Polynucleotide kinase from E.coli infected with T4 phageHas the reverse effect of alkaline phosphatase, adding phosphate groups onto free 5’
termini
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Terminal deoxynucleotidyl transferase from calf thymus tissue adds one or more deoxyribonucleotides onto the 3’ t
erminus of a DNA
DNA modifying enzymes
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Topoisomerases
Change the conformation of covalently closed-circular DNA by introducing or removing supercoils.
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Enzymes for cutting DNA-restriction endonucleases
The initial observation that led to the eventual discovery of restriction endonucleases (RE) was made in the early 1950s
Restriction occurs because the bacterium produces an enzyme (called restriction endonucleases) that degrades the phage DNA
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The discovery of these enzymes led to Nobel prizes for W.Arber, H. Smith and D. Nathans in 1978
Three different classes of RE are recognized, but the most important one is RE II which is used in DNA manipulation
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Type II restriction endonucleases (RE) cut DNA at specific nucleotide sequences
Generally, 4~8 bases be found, mostly 6 bases, a few of 8~10 bases
The sequences discriminated usually are palindrome structure
To cut the double strands of DNA at special sites and to yield two kinds of ends: blunt ends and sticky ends
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Blunt ends and sticky ends:
Sticky or cohesive ends:
the cleavage is staggered by two or four nucleotides
the resulting DNA fragments have short single-strand
ed overhangs at each end
Base pairing between them can stick the DNA molec
ule back together again
Restriction endonucleases with different recognition s
equences may produce the same sticky ends eg: Bam
H I (GGATCC) and Bgl II (AGATCT)
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5’-GGTGAATTCAGC…-3’3’-CCACTTAAGTCG…5’
5’-TTGCTGCAGAAG…-3’3’-AACGACGTCTTC…5’
5’-sticky end (EcoR I )
3’-sticky end ( Pst I )
5’-GGTG AATTCAGC…-3’3’-CCACTTAA GTCG…5’+
5’-TTGCTGCA GAAG…-3’3’-AACG ACGTCTTC…5’+
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blunt end or flush end
5’-CCCGGG…-3’3’-GGGCCC…5’
5’-CCC GGG…-3’3’-GGG CCC…5’+
*The ligation efficiency between the blunt ends is not as high as that of the stickly ends.
Sma I
Make a simple double-stranded cut in the middle of the recognition sequence
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Naming of RE
Escherichia coli RY13 I
EcoR I
The genus name of bacteria
The species name of bacteria
The strain name of bacteria
The order of the RE found in bacteria
REs are usually named after the bacterium from which they are isolated.
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The requirements for the gene cloning
(1) The target genes
(2) The vectors
(3) The tool enzymes
(4) The host cells
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The basic process of recombination technique
* The preparation of target DNA* The selection and preparation of vectors
* The ligation of DNA fragments in vitro
* Foreign DNA be transported into host cells
* The screening and identifying of target DNA
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1. The preparation of target DNA
(1)To prepare from genomic library genomic library contains a comprehensive DNA
fragments from genomic DNA cut by the specific RE.
During the construction of the genomic library, the DNA fragments and their vectors are ligated, and then introduced into the recipient cells.
It represents whole DNA sequence of a genome
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(2) To prepare from cDNA library or cDNA
Extracting total mRNA Reverse transcription
Ligation introduction
It represents the population of mRNAs coding for gene and protein expression
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(3) To prepare the gene fragment with
other methods
1) PCR amplification
2) To synthesize the DNA fragment by
chemical method
it is typically used for those of
the small biologically active peptides
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2. The selection and preparation of vectors
Plasmid λ phage cosmid M13 phage
Capacity < 10 kb < 22 kb 40~50 kb < 1 kbof cloning
gDNA library - + + -
cDNA library + + - -
Subcloning + - - +
Sequencing + + - +
E coli expression + + - -
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3. Construction of Recombinant Molecules
Both purified DNA fragments and vectors are digested with the same restriction enzyme to give complementary cohesive ends
•Analyzing the result of restriction endonuclease cleavage
Separation of molecules by gel electrophoresis Visualizing DNA molecules in a gel (EB staining) Comparison with size markers
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joining together of the vector molecules and DNA
to be cloned
The enzyme that catalyses the reaction is called D
NA ligase, which purified from E.coli bacteria that
have been infected with T4 phage
•Ligated by T4 ligase to recombinant molecules
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CTTAAGGAATTC
EcoR Ⅰ
CTTAAGGAATTC GAATTC5’CTTAAG 5’3’
3’ EcoR Ⅰ
CTTAAAATTC G5’
G 5’3’3’
GCTTAA
AATTCG
5’3’
5’ 3’
CTTAAG CTTAAGGAATTC GAATTC
ligationCTTAAG CTTAAGGAATTC GAATTC
Bidirection insertions
(1) Sticky-ended ligation
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VectorTarget gene
Restriction endonucleases
Restriction endonucleases
T4 DNAligase15ºC
recombinateSelf-ligated vector
Self-ligated target gene
(2) Blunt-ended ligation
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4. Introduction of DNA into living cells Serves two main purposes:
allows a large number of recombinant DNA molecule
s to be produced from a limited amount of starting ma
terial
Purification
-Methods Transformation
Transfection
Infection
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Transformation ----The uptake of DNA by bacterial cells preparation of competent E.coli cells
50 mM CaCl2 is tranditionally used.
Another alternative is by electroporation
• In recent years, transformation has been extended to include uptake of any DNA molecules by any type of cell
Whether the uptake results in a detectable change in the cell
Whether the Cells involved is bacterial, fungal, animal or plant
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Introduction of phage DNA into bacterial cells
Two methods:
Transfection
purified phage DNA, or recombinant phage molecules, is mixed with competent E.coli cells and DNA uptake induced by heat shock
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Transfection
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Introduction of phage DNA into bacterial cells Two methods:
In vitro packaging single strain system: the defective λphage c
arries a mutation in the cos sites two strain system: two defective λphage carr
ies a mutation in a gene for one of the components of the phage protein coat
Phage infection is visualized as plaques on the agar medium
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In vitro packaging
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The problem of selectionA restriction digest of total cell DNA produces not
only the fragment carrying the desired gene, but also many other fragments carrying all the other genes
Numerous different recombinant DNA molecules are produced
A variety of recombinant clones are obtained
5. Screening and Identification of Recombinants
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Target Genes Carried by Plasmid
1 plasmid1 cellRecombinant
PlasmidTransformation
Target GeneRecombination
Restriction
Enzyme
Restriction
Enzyme
Ch
rom
oso
mal
DN
ATarget Genes
DNA Recombination
TransformationHost Cells
Juang RH (2004) BCbasics
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Amplification and Screening of Target Gene
1
1 cell line, 1 colonyX100
X1,000
PlasmidDuplicationBacteria
Duplication
Plating
Pick the colonycontaining target gene
=100,000Juang RH (2004) BCbasics
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There are two basic strategies for obtaining the clone you want
Direct selection for the desired gene
the only clones that are obtained are clones of the required gene
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There are two basic strategies for obtaining the clone you want
Direct selection for the desired gene
the only clones that are obtained are clones of the required gene
Identification of the clone from a gene library
entails an initial shotgun cloning experiment, to produce a clone library representing all or most of the genes present in the cell, followed by analysis of the individual clones to identify the correct one
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Correct clone
A clone library
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Direct selection an antibiotic resistance gene
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Direct selection
an antibiotic resistance gene
Marker rescue ---by αcomplementation plasmids contain sequence (lacZ) coding
for N-terminal amino acids (α fragment) of β–galactosidase
Mutant cells contain sequence (lacZ) coding for C-terminal amino acids (ω fragment) of β– galactosidase
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By αcomplementationThe enzymatic activity is dependent on the
coexpression of the complete fragments, which can hydrolyzes the specific substrate X-gal (5-bromo-4-chloro-3-inolyl-β-D-galactoside) to turn to a blue colored one under the induction of IPTG ( isopropyl thiogalactoside)
The recombinant molecules have no this enzyme activity because the insertion of target gene into the lacZ region disturbs the expression of αfragment, and therefor, the colour of the recombinant molecule containing the colony is white.
Based on this blue-white colony screening
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White clone contains the recombinant, but blue clone not contain recombinant
Multiple cloning sites
The sequence coding the N end fragment of β-galactosidase
Ampr
promoter
transformation
Chromosome
The sequence coding the C end fragment of βgalactosidase
The growth of bacteria on the culture with X-gal
The blue clone containing the pUC18
The blue clone containing the pUC18
transformation
Cleavage N end
External DNA
Cleavage N end
Recombinant pUC18
The growth of bacteria on the culture with X-gal
The white clone containing the recombinant pUC18
α-mutual complement screening
Ampr
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Direct selection
an antibiotic resistance gene
Marker rescue ---by αcomplementation
Colony/plaque in situ hybridization is used for positive colony screening
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Colony Is Screened by Hybridization with Probe
Cover withfilter
paper
Autoradiography
Add probe
Transferring …
Collect filter paper
Dissolve cell DNA denatured
Jua
ng
RH
(2
00
4)
BC
ba
sics
Colony hybridization
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Direct selection
an antibiotic resistance gene
Marker rescue ---by αcomplementation
Colony/plaque in situ hybridization is used for positive colony screening
Immunological Technology
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Methods for clone identification
Colony PCREnzyme digestionnucleic acid hybridizationDNA sequencing
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Using PCR to detect gene targeting events
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M 1 2 3 4 5 6 7 8 9 10
1kb
Figure2b.The positive plasmids after cutted by EcoR IM----1Kb DNA ladder1∽10, positive plasmids cutted by EcoR
M 1 2 3 4 5 6 7 8 9 10
Figure2a. The clones of positive plasmidM----1Kb DNA ladder1∽10, positive plasmid
A. before cut with RE
To identify the target gene band after cutted by RE
B. after cut with RE
Identification with restriction enzymes
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The basic process of recombination technique
* The preparation of target DNA* The selection and preparation of vectors
* The ligation of DNA fragments in vitro
* Foreign DNA be transported into host cells
* The screening and identifying of target DNA
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6. Expression of the cloned gene
Different vectors are selected for cloning
cloning vectors are used for replicating and amplifying genes
Expression vector are applied to express the gene product
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Cloning vector
Antibiotics resistance, MCS and screening
Expression vector
Antibiotics resistance, MCS and screening
Contains regulatory sequences for transcription and translation, eg; promoter, SD sequence for 16s rRNA binding, and a terminator which is ρfactor independent
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Expression system
Prokaryote expression system E.coli (most popular) Its easy culture, fast proliferation, low
expense, large scale production Lack of the processing capability after
transcription and translation
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Notes during expression
Infusion proteins maybe formed when other sequences coding amino acids linked to a target gene are co-expressed together with target proteins
Purified by affinity chromatography, followed by the unnsecesary peptides cut out
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eukaryote expression system Mammalian cells are stable and
repeatable can process hnRNA to become mature
mRNA, as well as the post-translation
modifications
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Application of Recombination DNA Technology
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DNA Recombination Medical Production
产 品 功 能组织胞浆素原激活剂 抗凝血液因子 VIII 促进凝血颗粒细胞 - 巨噬细胞集落剌激因子 剌激白细胞生成促红细胞生成素 剌激白细胞生成生长因子 (bFGF, EGF) 刺激细胞生长与分化生长素 治疗侏儒症胰岛素 治疗糖尿病干扰素 ( 1b, 2a, 2b, ) 抗病毒感染及某些肿瘤白细胞介素 激活、剌激各类白细胞超氧化物歧化酶 抗组织损伤
单克隆抗体 利用其结合特异性进行诊断试验、肿瘤导向治疗
乙肝疫苗 (CHO, 酵母 ) 预防乙肝口服重组 B 亚单位菌体霍乱菌苗 预防霍乱
目 录
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Gene diagnosis It is recognized that the abnormal
structure and expression of the gene are involved in the pathogenesis of diseases
Gene diagnosis is the detection of the abnormalities of the candidate genes by ways of molecular biology and molecular genetics
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Sickle cell anemia belongs to gene point mutation The 6th codon GAG was changed to GTG Glu is changed to Val Abolish an MstII restriction site which spans codons 5-7
For examples :
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Sequencing HbS proteins revealed a single change: Glu6Val in the β chain. Fiber formation (R) at low [O2] causes sickling of RBCs (center).
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×
Mst restriction site (GCTNAGG)Ⅱ
5´ 3´
Normal gene
5´ 3´
Mutation gene
1.15kb
1.35kb
Restriction mapping analysis of sickle cell anemia
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++
﹣﹣
0.2kb
1.15kb
1.35kb
Normal Carrier Sickle cell homozygote
镰状红细胞贫血患者基因组的限制性酶切分析
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Gene Therapy Gene therapy is the way to transfer
genetic material which exerts the biological function into the cells of patients to treat the disease
Genetic material: normal gene, recombinant DNA, RNA, synthetic oligonucletides
They may integrate into the chromosome or express separately
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The Strategies and technologies of gene therapy
Gene correction The abnormal bases of a gene are correct
ed
Gene replacement The defected gene is replaced by the nor
mal one which can integrate into the chromosomes by homologous recombination or remain extrachromosomal
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Gene augmentation The target gene is introduced to the defecte
d cells or other cells
Gene inactivation the expression of the gene is intervened to b
lock or inhibit the inappropriate genes in vivo on both transcriptional and translational level
The Strategies and technologies of gene therapy
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Applications of Gene Therapy
The first apparently successful application was initiated on Sep. 14, 1990 for ADA deficiency
Results in a lymphopenic form of SCIResults in a lymphopenic form of SCID that is fatal in early childhood.D that is fatal in early childhood.
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ADA SCIDADA SCID ( (Severe Combined ImmunodeficiencySevere Combined Immunodeficiency DiseasesDiseases )
Autosomal recessive disorder ADA = adenosine deaminase (an enzyme
of purine metabolism) ADA is an important enzyme in the purinADA is an important enzyme in the purin
e catabolic pathway, catalyzing the irrevee catabolic pathway, catalyzing the irreversible deamination of adenosine to inosinrsible deamination of adenosine to inosine.e.
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SCID with ADA DeficiencySCID with ADA Deficiency
ADENOSINE
INOSINE
HYPOXANTHINE
XANTHINE
URIC ACID
GUANOSINE
GUANINE
Purine Catabolism Pathway
AdenosineDeaminase
PurineNucleoside
Phosphorylase
PurineNucleoside
Phosphorylase
XanthineOxidase
XanthineOxidase
Guanase
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SCID with ADA DeficiencySCID with ADA Deficiency
The enzyme deficiency inhibits the normal The enzyme deficiency inhibits the normal
catabolism of purines.catabolism of purines.
Results in the accumulation of metabolic suResults in the accumulation of metabolic su
bstrates that are toxic to lymphocytes, particbstrates that are toxic to lymphocytes, partic
ularly in the inhibition of lymphocyte functiularly in the inhibition of lymphocyte functi
on. on.
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GENE THERAPY IN ADA SCIDGENE THERAPY IN ADA SCID
ADA deficiency was the first disorder to be treated by gene therapy (Bordignon et al 1995)
The initial targets for genetic manipulation were bone marrow (BM) stem cellsbone marrow (BM) stem cells and peripheral blood lymphocytes (PBLs)peripheral blood lymphocytes (PBLs)
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Vectors expressing human ADAcDNA (1.5 kbp) with their own promoters were transfected into BM stem cellsBM stem cells and PBLsPBLs in vitro
6 months after gene therapy ended, vector-derived DNA was found in the PBLsPBLs
GENE THERAPY IN ADA SCIDGENE THERAPY IN ADA SCID
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The Cloning Procedure Used for Creating Dolly