Gene cloning,expression
and functional study
基因克隆，表达及功能研究
vectors
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
through a vector
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 resistance
blue-white screening
3,Contains a multiple cloning site (MCS)
4,Easy to be isolated from the host cell.
A plasmid vector for cloning
Ampicillin resistant? yes yes
Tetracycline resistant? No yes
B X B
B
B
X
Ampr
ori
Ampr
Tcr
ori
pBR322
Ampr Tcr
ori
-Screening by insertional inactivation of a resistance gene
Twin 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 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 transformants
Recombinant plasmid containing inserted DNA,
white transformants
Recreated vector (no insert)
Recombinant plasmid (contain insert)
back
Multiple cloning sites
Multiple 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 KpnI
SmaI
XmaI BamHI XbaI
SalIHincII
AccI PstI SphI
Lac Z
A 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
RBS
Start codon
MCS
Transcription
terminator
Ampr
ori
T7
expression
vector
An bacterial expression vector
MCS
A yeast expression 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 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 fragments
(Eukaryotic Genome project)
Genomic library VS cDNA library
Cosmid 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 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 YAC
2,Capacity is 300-350 kb
3,One to two copies in each cell
4,Easy to handle
5,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 × 105ln(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 selectivity
BamH1,5’-G/GATCC
Selection of restriction enzyme
1,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 libraries
I2-1 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
are 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
expression 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 translated
Analysis 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 gels
Enrichment,carried out by hybridization
Example,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 transcriptase
Four dNTPs
AAAAA-3’
TTTTTP-5’
mRNA
mRNA
cDNA
cDNA
cDNA
Duplex cDNA
AAAAA-3’
TTTTTP-5’
TTTTTP-5’
3’
3’-CCCCCCC
Terminal transferase
dCTP
Alkali (hydrolyaes RNA)
Purify DNA oligo(dG)
Klenow polymerase or reverse
Transcriotase Four dNTPs
5’-pGGGG-OH
5’
3’-CCCCCCC
5’-pGGGG
3’-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 ends
Blunt 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 suitable
for cloning the cDNA.
The process,
Dephosphorylate the vector with alkaline
phosphatase
Ligate vector and cDNA with T4 DNA ligase
(plasmid or λ phage vector)
I2 cDNA libraries
I3 Screening procedures
I3-1 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 gene
Probes:
? 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 a
Nylon 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 or
repeated hybridization
(Alkali treatment)
I3 Screening procedures
Transfer to nitrocellulose
or 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 activity
2,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
and functional study
基因克隆，表达及功能研究
vectors
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
through a vector
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 resistance
blue-white screening
3,Contains a multiple cloning site (MCS)
4,Easy to be isolated from the host cell.
A plasmid vector for cloning
Ampicillin resistant? yes yes
Tetracycline resistant? No yes
B X B
B
B
X
Ampr
ori
Ampr
Tcr
ori
pBR322
Ampr Tcr
ori
-Screening by insertional inactivation of a resistance gene
Twin 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 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 transformants
Recombinant plasmid containing inserted DNA,
white transformants
Recreated vector (no insert)
Recombinant plasmid (contain insert)
back
Multiple cloning sites
Multiple 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 KpnI
SmaI
XmaI BamHI XbaI
SalIHincII
AccI PstI SphI
Lac Z
A 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
RBS
Start codon
MCS
Transcription
terminator
Ampr
ori
T7
expression
vector
An bacterial expression vector
MCS
A yeast expression 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 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 fragments
(Eukaryotic Genome project)
Genomic library VS cDNA library
Cosmid 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 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 YAC
2,Capacity is 300-350 kb
3,One to two copies in each cell
4,Easy to handle
5,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 × 105ln(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 selectivity
BamH1,5’-G/GATCC
Selection of restriction enzyme
1,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 libraries
I2-1 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
are 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
expression 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 translated
Analysis 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 gels
Enrichment,carried out by hybridization
Example,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 transcriptase
Four dNTPs
AAAAA-3’
TTTTTP-5’
mRNA
mRNA
cDNA
cDNA
cDNA
Duplex cDNA
AAAAA-3’
TTTTTP-5’
TTTTTP-5’
3’
3’-CCCCCCC
Terminal transferase
dCTP
Alkali (hydrolyaes RNA)
Purify DNA oligo(dG)
Klenow polymerase or reverse
Transcriotase Four dNTPs
5’-pGGGG-OH
5’
3’-CCCCCCC
5’-pGGGG
3’-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 ends
Blunt 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 suitable
for cloning the cDNA.
The process,
Dephosphorylate the vector with alkaline
phosphatase
Ligate vector and cDNA with T4 DNA ligase
(plasmid or λ phage vector)
I2 cDNA libraries
I3 Screening procedures
I3-1 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 gene
Probes:
? 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 a
Nylon 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 or
repeated hybridization
(Alkali treatment)
I3 Screening procedures
Transfer to nitrocellulose
or 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 activity
2,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
