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Gene cloning, expression
and functional study
基因克隆,表达及功能研究
vectorsvectors
Cloning vectors: 克隆载体to clone a gene in a vector
Expression vectors: 表达载体to express a gene from a vector
Integration vectors: 整合载体to integrate a gene in a genome thr
ough a vector
Cloning vectors: 克隆载体to clone a gene in a vector
Expression vectors: 表达载体to express a gene from a vector
Integration vectors: 整合载体to integrate a gene in a genome thr
ough a vector
Cloning vectors Cloning vectors
1 Plasmid vecters
2 Bacteriophage vectors
3 Cosmids & BACs
4 Eukaryotic vectors
Cloning vectors: allowing the exogenous DNA to be inserted, stored, and manipulated mainly at DNA level.
expression vectors: allowing the exogenous DNA to be inserted, stored, and expressed.
1. Contains an origin of replication, allowing for replication independent of host’s genome.
2. Contains Selective markers: Selection of cells containing a plasmid twin antibiotic resistanceblue-white screening
3. Contains a multiple cloning site (MCS)4. Easy to be isolated from the host cell.
A plasmid vector for cloning A plasmid vector for cloning
Ampicillin resistant? yes yesTetracycline resistant? No yes
B X B
B
B
XAmpr
ori
Ampr
Tcr
ori
pBR322
Ampr Tcr
ori
-Screening by insertional inactivation of a resistance geneTwin antibiotic resistance screeningTwin antibiotic resistance screening
Replica plating: transfer of the colonies from one plate to another using absorbent pad or Velvet ( 绒布 ).
transfer of colonies
+ampicillin + ampicillin+ tetracycline
these colonies have bacteria with recombinant plasmid
Blue white screeningBlue white screening
Ampr
ori
pUC18(3 kb)
MCS (Multiple cloning sites,多克隆位点)
Lac promoter
lacZ’
Screening by insertional inactivation of the lacZ gene
The insertion of a DNA fragment interrupts the ORF of lacZ’ gene, resulting in non-functional gene product that can not digest its substrate x-gal.
Recreated vector: blue transformantsRecombinant plasmid containing inserted DNA: white transformants
Recreated vector (no insert)
Recombinant plasmid (contain insert)
back
Multiple cloning sitesMultiple cloning sitesMultiple restriction sites enable the convenient insertion of target DNA into a vector
Ampr
ori
pUC18(3 kb)
MCS (Multiple cloning sites,多克隆位点)
Lac promoter
lacZ’
…ACGAATTCGAGCTCGGTACCCGGGGATCCTCTAGAGTCGACCTGCAGGCATGCA…
. T h rA s n S er S e r Val Pro Gly Asp Pro Leu Glu Ser Thr Cys Arg His Ala Ser…
EcoRI SacI KpnISmaIXmaI BamHI XbaI
SalIHincIIAccI PstI SphI
Lac Z
A plasmid vector for gene expressionA plasmid vector for gene expression
Expression vectors: allowing the exogenous DNA to be inserted, stored and expressed.
1. Promoter and terminator for RNA transcription are required.
2. Intact ORF and ribosomal binding sites (RBS) are required for translation.
3. Include : (1) bacterial expression vectors, (2) yeast expression vectors, (3) mammalian expression vector
T7 promoter
RBSStart codon
MCS
Transcription terminator
Ampr
ori
T7 expression
vector
An bacterial expression vectorAn bacterial expression vector
MCS
A yeast expression vectorA yeast expression vector
Bacteriophage vector Bacteriophage vector
Two examples:1.λ phage • bacteriophageλ• λ replacement vector 2.M13 phage• M13 phage vector• Cloning in M13• Hybrid plasmid-M13 vectors
• viruses that can infect bacteria. •48.5 kb in length•Linear or circular genome (cos ends)•Lytic phase (Replicate and release)•Lysogenic phase (integrate into host genome)
λ phageλ phage
Analysis of eukaryotic genes and the genome organization of eukaryotes requires vectors with a larger capacity for cloned DNA than plasmids or phage .
Human genome (3 x 109 bp): large genome and large gene demand vectors with a large size capacity.
Cloning large DNA fragmentsCloning large DNA fragments(Eukaryotic Genome project)
Genomic library VS cDNA libraryGenomic library VS cDNA library
Cosmid vectorsCosmid vectors
1. Utilizing the properties of the phage cos sites in a plasmid vector.
2. A combination of the plasmid vector and the COS site which allows the target DNA to be inserted into the head.
3. The insert can be 37-52 kb
Digestion
Ligation
C) Packaging and infect
Formation of a cosmid clone
YAC vectorsYAC vectors
Accommodates genomic DNA fragments of more than 1 Mb, and can be used to clone the entire human genome, but not good in mapping and analysis.
(yeast artificial chromosome)
Essential components of YAC vectors :• Centromers (CEN), telomeres (TEL) and autonomous replicating sequence (ARS) for proliferation in the host cell. • ampr for selective amplification and markers such as TRP1 and URA3 for identifying cells containing the YAC vector in yeast cells. • Recognition sites of restriction enzymes (e.g., EcoRI and BamHI)
YAC Cloning
BAC vectors 细菌人工染色体
1. More stable than YAC2. Capacity is 300-350 kb3. One to two copies in each cell4. Easy to handle5. More popular in genomic mapping
I1 Genomic libraries
I1-1 Representative gene libraries
I1-2 Size of library I1-3 Genomic DNA I1-4 Vectors
Gene libraries and screening
Gene library: a collection of different DNA sequence from an organism, each of which has been cloned into a vector for ease of purification, storage and analysis.
Genomic libraries
cDNA libraries
Gene library (made from genomic DNA)
(made from cDNA- copy of mRNA)
I1 Genomic
libraries
I1-1 Representative gene libraries
--- Contain all the original sequences
1. Certain sequences have not been cloned.Example: repetitive sequences lacking restriction sites
2. Library does not contain sufficient clones
Missing original sequence
Too long for the vector used
I1 Genomic
libraries
I1-2 Size of library (ensure enough clones)
must contain a certain number of recombinants for there to be a high probability of it containing any particular sequence The formula to calculate the number of recombinants:
N = ln (1-P)
ln (1-f)
P: desired probability f : the fraction of the genome in one insert
I1 Genomic
libraries
For example :for a probability of 0.99 with insert sizes of 20 kb these values for the E.coli (4.6×106 bp) and human (3×109 bp) genomes are :
N E.coli= = 1.1 ×103 ln( 1-0.99)
ln[1-(2×104/4.6×106)]
Nhuman= = 6.9 ×105 ln(1-0.99)
ln[1-(2 ×104/3 ×109)]These values explain why it is possible to make good genomic libraries from prokaryotes in plasmids where the insert size is 5-10kb ,as only a few thousand recombinants will be needed.
I1 Genomic
libraries
I1-3 Genomic DNA libraries
Purify genomic DNA
Fragment this DNA : physical
shearing and restriction enzyme
digestion
eukaryotes
prokaryotes
Clone the fragments into vectors
I1 Genomic
libraries
To make a representative genomic libraries ,genomic DNA must be purified and then broken randomly into fragments that are correct in size for cloning into the chosen vector.
Purification of genomic DNA :
Prokaryotes :extracted DNA directly from cells
remove protein, lipids and other unwanted macro-molecules by protease digestion and phase extraction.
Eukaryotes :prepare cell nuclei
I1 Genomic
libraries
Break DNA into fragments randomly:
Physical shearing : pipeting, mixing or sonicaion
Restriction enzyme digestion: partial digestion is
preferred to get a greater lengths of DNA fragments.
I1 Genomic
libraries
Sau3A: 5’-/GATC-3’, less selectivityBamH1: 5’-G/GATCC
Selection of restriction enzyme1. Ends produced (sticky or blunt) &
The cleaved ends of the vector to be cloned
2. Whether the enzyme is inhibited by DNA modifications (CpG methylation in mammals
3. Time of digestion and ratio of restriction enzyme to DNA is dependent on the desired insert size range.
I1 Genomic
libraries
I1-4 Vectors
According to genome’s size,we can select a proper vector to construct a library .
Vectors Plasmid phageλ cosmid YAC insert (kb) 5 23 45 1000
The most commonly chosen genomic cloning vectors are λ relacement vectors which must be digested with restriction enzymes to produce the two λ end fragment or λ arms between which the genomic DNA will be digested
I1 Genomic
libraries
cos cos
Long (left)arm
short (right)arm
Exogenous DNA(~20-23 kb)
λ phage vector in cloning
cos cos
Long (left)arm
short (right)arm
Exogenous DNA(~20-23 kb)
λ replacement vector cloning
2. Packing with a mixture of the phage coat proteins and phage DNA-processing enzymes 3. Infection and
formation of plaques
Library constructed
1. Ligation
0.preparation of arm and genomic inserts
I 2 cDNA librariesI2-1 mRNA isolation, purification
I2-2 Check theRNA integrity
I2-3 Fractionate and enrich mRNA
I2-4 Synthesis of cDNA
I2-5 Treatment of cDNA ends
I2-6 Ligation to vector
Gene libraries and screening
cDNA libraries
1.No cDNA library was made from prokaryotic mRNA.
• Prokaryotic mRNA is very unstable• Genomic libraries of prokaryotes ar
e easier to make and contain all the genome sequences.
I 2 cDNA libraries
2.cDNA libraries are very useful for eukaryotic gene analysis
• Condensed protein encoded gene libraries, have much less junk sequences.
• cDNAs have no introns genes can be expressed in E. coli directly
• Are very useful to identify new genes• Tissue or cell type specific (differential ex
pression of genes)
cDNA libraries
I 2 cDNA libraries
I2-1 mRNA isolation
• Most eukaryotic mRNAs are polyadenylated at their 3’ ends
• oligo (dT) can be bound to the poly(A) tail and used to recover the mRNA.
AAAAAAAAAAn5’ cap
I 2 cDNA libraries
I 2 cDNA libraries
1.Traditionally method was done by pass a preparation of total RNA down a column of oligo (dT)-cellulose
2.More rapid procedure is to add oligo(dT) linked to magnetic beads directly to a cell lysate and ‘pulling out’ the mRNA using a strong magnet 3.Alternative route of isolating mRNA is lysing cells and then preparing mRNA-ribosome complexes on sucrose gradients
Three methods to isolate mRNA.
I2 cDNA libraries
Make sure that the mRNA is not degraded. Methods:Translating the mRNA : use cell-free translation system as wheat germ extract or rabbit reticulocyte lysate to see if the mRNAs can be translatedAnalysis the mRNAs by gel elctrophoresis: use agarose or polyacrylamide gels
I2-2 Check the mRNA integrity
I2 cDNA libraries
I2-3 Cloning the particular mRNAs
Is useful especially one is trying to clone a particular gene rather to make a complete cDNA library.
Fractionate on the gel: performed on the basis of size, mRNAs of the interested sizes are recovered from agarose gelsEnrichment: carried out by hybridizationExample: clone the hormone induced mRNAs (substrated cDNA library)
I2 cDNA libraries
I2-4 Synthesis of cDNA :
First stand synthesis: materials as reverse transcriptase ,primer( oligo(dT) or hexanucleotides) and dNTPs (Fig 1.1)
Second strand synthesis: best way of making full-length cDNA is to ‘tail’ the 3’-end of the first strand and then use a complementary primer
to make the second. (Fig2.1)
I2 cDNA libraries
5’ mRNA AAAAA-3’ HO-TTTTTP-5’
5’
Reverse transcriptaseFour dNTPs
AAAAA-3’TTTTTP-5’
mRNA
mRNA
cDNA
cDNA
cDNA
Duplex cDNA
AAAAA-3’
TTTTTP-5’
TTTTTP-5’
3’
3’-CCCCCCC
Terminal transferasedCTP
Alkali (hydrolyaes RNA)Purify DNA oligo(dG)
Klenow polymerase or reverseTranscriotase Four dNTPs
5’-pGGGG-OH
5’
3’-CCCCCCC
5’-pGGGG3’-CCCCCCC TTTTTP-5’
-3’
Fig 1.1 The first strand synthesis
I2 cDNA libraries
5’-pGGGG3’-CCCCCCC
HO-CCGAATTCGGGGGG 3’-GGCTTAAGCCCCCC
5’-pAATTCGGGGGG
TTTTTGGCTTAAGCC-OH CCGAATTCGG-3’
3’-CCCC
3’-CCCCCCC
3’-CCC 5’-pGGGG
5’-pGGGG
TTTTTp-5’ -3’
TTTTTp-5’
TTTTTp-5’
-3’
-3’
TTTTTGGCTTAAp-5’
HO-CCG/AATTCGG-3’ 3’-GGCTTAA/GCC-OH
CCG-3’
Duplex cDNA
Single strand-specific nuclease
Klenow polymerase
treat with E.coRI methylase
Add E.colRI linkers using T4 DNA ligase
E.colRI digestion
Ligate to vector and transfom
Fig2.1 Second strand synthesis
I2-5 Treatment of cDNA endsBlunt and ligation of large fragment is not efficient, so we have to use special acid linkers to create sticky ends for cloning.
The process :
Move protruding 3’-ends(strand-special nuclease)
Fill in missing 3’ nucleotide (klenow fragment of DNA polyI and 4 dNTPs)
Ligate the blunt-end and linkers(T4 DNA ligase)
Restriction enzyme digestion (E.coRI )
Tailing with terminal transferase or using adaptor molecules
I2 cDNA libraries
I2-6 Ligation to vector Any vectors with an E.coRI site would suitablefor cloning the cDNA.
The process :
Dephosphorylate the vector with alkalinephosphatase
Ligate vector and cDNA with T4 DNA ligase
(plasmid or λ phage vector)
I2 cDNA libraries
I3 Screening proceduresI3-1 Screening
I3-2 Colony and plaque hybridization
I3-3 Expression screening
I3-4 Hybrid arrest and release
I3-5 Chromosome walking (repeat screening)
Gene libraries and screening
I3-1 Screening
The process of identifying one particular clone containing the gene of interest from among the very large number of others in the gene library .
1. Using nucleic acid probe to screen the library based on hybridization with nucleic acids.
2. Analyze the protein product.
I3 Screening procedures
Screening libraries
Hybridization to identify the interested DNA or its RNA product
1. Radiolabeled probes which is complementary to a region of the interested geneProbes: • An oligonucleotide derived from the
sequence of a protein product of the gene• A DNA fragment/oligo from a related gene
of another species 2. Blotting the DNA or RNA on a membrane 3. Hybridize the labeled probe with DNA
membrane (Southern) or RNA (Northern) membrane
Searching the genes of interest in a DNA library
I3 Screening procedures
I3-2 Colony and plaque hybridization
Transfer the DNA in the plaque or colony to aNylon or nitrocellulose membrane
Phage DNA bind to the membrane directly
Bacterial colonies must be lysed to release DNA on the membrane surface.
Hybridization (in a solution Containing Nucleic acid probe)
Wash to remove unhybri-dization probe and visualize
X-ray film(radio-actively labeled )
antibody or enzyme(modified nucleotide labeled
Line up the hybridizated region orrepeated hybridization
(Alkali treatment)
I3 Screening procedures
Transfer to nitrocelluloseor nylon membrane
Denature DNA(NaOH)Bake onto membrane
Probe with 32p-labled DNA complementary to gene of interest
Expose to film
Select positive from master plate
Keep master plate
Screening by plaque hybridization
I3 Screening procedures
Identify the protein product of an interested gene
1.Protein activity2.Western blotting using a
specific antibody
I3 Screening procedures
I3-3 Expression screening
Expression screening (1)
If the inserts are cloned into an expression sites, it may be expressed. Therefore, we can screen for the expressed proteins. However, this screening may miss the right clone
Example: the EcoRI site of lgt11 vector. The inserted genes have one in six change (1/6) to be in both the correct orientation (2 possibilities; ) and reading frame (three possibilities; three nucleotide code XXX).
I3 Screening procedures
Expression screening (2)
The procedure has similarities to the plaque hybridization protocol.
‘Plaque lift’ ( taken by placing a membrane on the dish of plaque)
Immersed in a solution of the antibody
Detected by other antibodies
Repeat cycles of screening to isolate pure plaques
Antibodies can be used to screen the expression library.
I3 Screening procedures
基因表达
1. Prokaryotic expression vector
原核表达载体2. Baculovirus expression vector
昆虫杆状病毒表达载体3. Mammalian expression vector
哺乳动物表达载体4. Adenoviral and retroviral vector
腺病毒及逆转录病毒表达载体
Prokaryotic expression vector
原核表达载体
GST-fusion
6xHis-fusion
GST
HIS
基因功能研究
1. Overexpression in cells 超表达,观察表型2. RNAi 干扰技术3. Yeast two hybrid system 酵母双杂交等技术
寻找与目的基因相关的蛋白4. Protein expression and antibody preparation
表达蛋白与抗体制备5. Localization of protein 蛋白在细胞中的定位
The end