recombinant DNA technology (genetic engineering) · 2018-11-12 · recombinant DNA technology...
Transcript of recombinant DNA technology (genetic engineering) · 2018-11-12 · recombinant DNA technology...
Chapter 23
recombinant DNA technology
(genetic engineering)
授课老师:陈蔚文
山东大学医学院生物化学与分子生物学系
SetcionⅠ
DNA Recombination and Gene Transfer
Occur Frequently in Nature
DNA recombination
conjugation
transformation
transduction
transposition
homologous recombination
site-specific recombination
SetcionⅡ
DNA Recombination Technique
DNA Cloning
Molecular Cloning
Genetic engineering
5
Naissance of gene engineering
Theory basis:
•1865, G.J.Mendel
•1944, O.T. Avery
•1953, double helix
•1961, control of gene expression
Technology basis:
•Small molecular vector: plasmid, virus, phage
•Restriction endonuclease: cut DNA in vitro
•DNA ligase: ligate DNA in vitro
6
DNA Stores Genetic Information
replication replication
transcription transcription
translationtranslation
Recombinant DNA Technology
8
History
•1972, Boyer, EcoRⅠ
•1972, P. Berg, SV40 & λ phage were cut with EcoRⅠ,
then ligated with T4 DNA ligase — first recombinant
experiment in vitro, 1980 Nobel prize
•1973, Cohen, R6-5 (Kan+) & pSC101 (tet+) were cut
with EcoRⅠ and then ligated with T4 DNA ligase,
resisted kan and tet.
9
1974, Cohen & Boyer, a gene from Xenopus laevis was recombined with pSC101 and transformed into E. coli, the mRNA of this gene was detected into E. coli —first gene clone experiment
1974, P. Berg , NIH, Recombinant DNA Advisory Committee
1976, NIH, rule for Recombinant DNA study
1977, Boyer founded Genentech with venture capitalist Robert A. Swanson.
1997, Cloned sheep Dolly
Concept: DNA cloning
Materials:
– Enzymes
– Target DNA
– Vectors
– Host cells
Basic strategy
Content:
Clone
A population of identical cells or DNA
molecules descended from a single
progenitor. Viruses or organisms that
are genetically identical and
descended from a single progenitor.
In vitro, attach the target DNA fragments to the
genetic elements that can autonomously
replicating (carrier DNA) , to form recombinant
DNA molecules, and then introduced into
recipient cell to replicate and amplify, resulting
in a large number copies of a single DNA
molecule.
DNA Cloning
Definition:
Recombinant DNA/Gene cloning /DNA cloning
With some methods or
techniques recombinant /
chimeric DNA molecules are
formed by joining different
DNA fragments from different
sources in vitro.
The recombinant DNA molecules
are introduced into appropriate
host cells in which they can be
replicated or expressed. The host
cells can reproduce and all of its
descendants carrying the same
recombinant DNA molecules are
called a clone. So the
recombinant DNA is cloned by
this process.
Sch
emati
c il
lust
rati
on
of
DN
A c
lon
ing
18
Materials and tools for DNA cloning include:
Target DNA: DNA fragment or gene you want
to study
Vehicle: Plasmids or viral vectors
Scissors: Restriction enzymes
Glue: DNA ligase
Host cells:prokaryotes or eukaryotes
19
Ⅰ Tool enzyme
1. Restriction Endonucleases
Restriction endonuclease : Site-specific endodeoxyribonucleases that
cleave both strands of DNA at points in or near
the specific site recognized by the enzyme;
important tools in genetic engineering.
Restriction-modification system
in bacteria
restriction endonuclease and methylase
restriction endonuclease can cleave foreign DNA;
“restrict” the entry of DNA, protect bacteria own
DNA
3681 kinds restriction endonuclease, type I, II and III
22
Restriction endonuclease Ⅱ
Cut DNA sequence-specifically;
Named (e.g., EcoRI) for bacterial genus,
species, strain, and type;
Recognize specific 4~8 bp sequences
that have symmetry (palindrome);
The cut ends are blunt or sticky/
cohesive ends;
A key tool in recombinant DNA research.
Restriction-modification system
24
The interaction of EcoRI endonuclease with its target sequence.
RE
Phosphodiester bond
Restriction enzymes are named after the
bacterium from which they are isolated.
eg. EcoR I
The first letter
of the genus
the first two letters
of the species
The first letter
of the strain
The order of
discovery
The endonucleases recognize and cleave a specific double-stranded DNA sequence that is 4~8bp long sequence.
sticky end
blunt end
The DNA cuts produced by restriction enzymes have two types of ends depending on the cut position by the enzymes.
EcoRI
PstI5'
3'
3'
-G ACGTC-
-CTGCA G-
5'
5' 3'-G AATTC-
5'3' -CTTAA G-
3' sticky end
5' sticky end
Sticky ends are particular useful in constructing
recombinant or chimeric DNA molecules.
32
Using the same enzyme to cut DNA molecules from different sources is most useful in recombinant DNA technology.
intermolecular ligation
GGATCC
CCTAGG
G
CCTAG
GATCC
G
BamHI
GGATCCCCTAGG
GCCTAG
GATCCG+
Bam HⅠ
GGATCCCCTAGG
GCCTAG
GATCCG+
BstⅠ
Isoschizomer are pairs of restriction enzymes specific to the same recognition sequence, isolated from different strains of bacteria and therefore may require different reaction conditions
BamH Ⅰ
Sau3A Ⅰ
5' 3'-G GATCC-
5'3' -CCTAG G-
5'
5'3'
3'-N GATCN-
-NCTAG N-
Compatible end
Isocaudomers are pairs of restriction enzymes
that have slightly different recognition sequences
but upon cleavage generate identical termini
2.DNA Ligase: another important and useful
enzyme in recombinant DNA technology.
37
DNA ligase can join together any two DNA fragments
in vitro to produce recombinant DNA molecules.
The insertion of a DNA fragment into a bacterial
plasmid with the enzyme DNA ligase
nick
gap
Nick
Ⅱ Target DNA interest
Genomic DNA
cDNA
41
Ⅲ Vector DNA
42
Vector DNA is a DNA molecule capable of self-replication
in a host organism and into which a piece of
DNA can be inserted, including cloning vector
and expressing vector.
Self-replication in host cells: the origin for
replication is required for the vector to
propagate.
Several unique recognition sequences for
different restriction enzymes that can provide
sites where the vector can be cut and foreign
DNA fragment can be inserted.
Marks for selection of the cells that contain the
vector or recombinant vector
1. Cloning vector
① Cloning vectors must have 3 basic features
The constructed
E. coli plasmid
pBR322.
insert
replication
BamHI
②Plasmids:
small, circular, duplex DNA molecule ususally1~200kb size.
exist in the cytosol of the bacterium, symbiotic with the host.
replicate independently of the bacterial DNA.
confer antibiotic resistance to the host cell.
many constructed plasmids are available commercially.
their complete sequences are known.
some nonessential sequences are removed.
synthetic polylinker with unique sites forseveral restriction enzymes is inserted.
47
48
49
Some of the important features of pBR322
Several unique recognition sequences for different restriction endonucleases provide sites where the plasmid can be cut and foreign DNA inserted.
Have a origin of replication ( ori )
Ampicilin resistance gene (ampr )、tetracycline resistance gene ( tetr ) allow the selection of cells
An overall small size which can facilitate the plasmid entry into cells.
Multiple cloning
site (MCS)
52
1.Lac operon structure
regulatory region
coding region
Z:β-galactosidase
Y: permease
A: transacetylase
promoter
operator
lac operon
CAP binding site
54
α-complementation
Some plasmid vectors such as pUC19 carry the lac Z’ gene that express α fragment of β-galactosidase. The α fragment is the N-terminus of the β-galactosidase. Typically, the mutant E. coli host strain only carry the ω fragment, which is the C-terminus of the protein. Either ω omega or α fragment alone is nonfunctional. When the vector containing lac Z’ introduced into mutant E. coli, both the α and ω fragments are present there is an interaction and a functionally intact beta-galactosidase protein can be produced. This interaction is called α complementation
56
There are two categories of plasmids
Stringent plasmids:replicate only
when the chromosome replicates. 1~2
Relaxed plasmids:replicate on their
own. This gives you a higher ratio of
plasmids to chromosome. ≥10
Other cloning vectors
Lambda phage: λgt & EMBL
M13 phage: M13mp, pUC
Cosmid (cos site-carrying plasmid )
BAC (Bacterial Artificial Chromosomes )
YAC (Yeast artificial chromosome)
Vector Host Characteristics Insert size range
Plasmid bacteria, yeast Small circular DNA <5 - 10 kb
phage λ bacteria Linear viral DNA up to ~20 kb
Cosmid bacteria Hybrid of plasmid and phage up to ~50
BAC bacteria Small circular DN 300kb
YAC yeast DNA containing yeast centromere,
telomeresand origins of replication
~200 to ~1000 kb,
<2Mb
Vectors used in molecular cloning
Bacterial artificial
chromosomes (BACs) as
cloning vectors.
Con
stru
ctio
n o
f a y
east
art
ific
ial
chro
moso
me
(YA
C)
Cloning Vectors must contain:
Origin of replication: DNA polymerase
Selectable marker(s): antibiotic resistance
Multiple cloning sites (restriction enzyme
sites): cutting/pasting of DNA fragments
2. Expression vector: Cloning vectors with the transcription and translation signals
Promoter: recognized by RNA polymerase
Production of large
amounts of a protein
from a protein coding
DNA sequence cloned
into an expression
vector and introduced
into cells.
①D
NA
se
qu
en
ce
s i
n a
typ
ica
l
E. co
li e
xp
res
sio
n v
ecto
r
Regulated expression
of RecA protein in a
bacterial cell.
70
② Eukaryotic expression vector
Shuttle vector
Eukaryotic expression regulatory
elements
Yeast, insect, and mammal
Shuttle Vectors Are Plasmids That Can Propagate in Two Different Organisms
ⅣHost cells:
1.easy to take up the recombinant DNA.
2.no selective markers that the vector contained.
3.no restriction for the replication of the
recombinant DNA.
4.no restriction enzymes that can cut the
recombinant DNA.
5.they are prokaryotes or eukaryotes
73
ⅤCloning strategy
Separation: A specific gene or DNA fragment and
vector DNA you chosed are separated from their originals
Cut: Vector DNA and target DNA are cut at precise
locations by proper restriction enzymes.
Ligation: The target DNA is linked to the vector DNA
covalently by DNA ligase to form a recombinant DNA.
Transformation: The recombinant DNA is introduced
into a host cell that can provide the enzymatic machinery
for DNA replication.
Selection or screening: the host cells that contain the
recombinant DNA are identified by using some methods.
Cloning gene expression: proteins expression
75
The procedures of gene cloning
Separation
Separating a specific gene or DNA fragment from its original as
target gene that you studied or you are interested in.
Acquirement of target gene:
(1). Separated directly from chromosomal DNA, easily in
prokaryotes.
digestion with restriction enzyme
separated by electrophoresis_
+
markers
samples
77
(2) Artificial synthesis of simple and
short sequence.
working backward from the amino
acid sequence, deducing the DNA
sequence that would code for it
obtain the necessary DNA sequence
information from sequence databases
(3) Selection from gene library
Gene library: a collection of DNA clones, gathered
together as a source of DNA for sequencing, gene
discovery, or gene function studies. Include
genomic DNA library and cDNA library.
Genomic DNA library: A DNA library containing
DNA segments that represent all (or most) of
the sequences in an organism’s genome.
cDNA library: DNA library consisting entirely of
cloned cDNAs from a particular organism or cell
type under certain conditions.
80
The vector for construction of library
λ Phage
Cosmid
BAC: Bacterial artificial chromosome
YAC: Yeast artificial chromosome
*Phage λ DNA
- a bacteriophage that can
infect E.coli.
-linear double-stands DNA,
48502 bp in length.
-self-replication in host cell
The genome of λ phage consist of left arm, central portion and right arm. Both arms are required by lytic growth, but central portion is not essential for replication of λ DNA and can be broken or removed.
central portion (~15 kb)
Insertion /replacement vectors
DNA is packaged into infectious phage particles
only if it is between 40~53 kb long
Insertion vectors:
Foreign DNA sequence is inserted into
the λ genome without any significant change
of the wild type genome.
Smaller insert size (up to ~10kb).
They may contain a multiple cloning site
inserted in lacZ system for screening of
recombinant bacterial colonies.
Can be used to clone smaller DNA molecule.
Such as λ ZAP, λ gt, etc.
84
Replacement vectors:
Full length λ molecule having two identical
restriction sites flanked by “stuffer fragment”.
Stuffer fragment is replaced by foreign DNAduring restriction cloning.
The vector without the foreign insert cannot bepackaged due to the size limitation (smallerthan the required).
Insert size ranges between 10-23 kb.
Eg. λ EMBL 3, λ EMBL 4, λ DASH etc.
86
Genomic Library
a collection of bacteria (or bacteriophage) that contain all the DNA in the organism’s genome on plasmids; every gene in the entire genome is present in the genomic library
BamH Ⅰ 5' 3'-G GATCC-
5'3' -CCTAG G-
Sau3A Ⅰ5'
5'3'
3'-N GATCN-
-NCTAG N-
Replacement vector
89
cDNA library
90
In construction of cDNA libraries we make use of
the fact that all translated mRNAs in eukaryotic
cells contain poly (A) tail.
91
The
synth
esis
of
cDN
A.
94
95
cDNAs can
be introduced
into a
cloning /
expression
vector and
cloned in a
bacterial host
cDNA
The
dif
fere
nce
s b
etw
een c
DN
A c
lones
and g
eno
mic
DN
A c
lones
der
ived
fro
m
the
sam
e re
gio
n o
f D
NA
Clone everything you can and then find what you need.
How to clone a specific gene from gene library?
How to find the clone with "our" gene?
The most common methods include:
1. Phenotypic screening
2. DNA hybridization
3. Screening with antibodies
Phenotypic screening is used when cloned gene is
expressed and changes properties of the cell in an
"obvious way". In the shown example, the protein
encoded in the cloned gene changes the color of
transformed cells.
DNA hybridization
Use o
f hybridiz
ation to identify
a c
lone w
ith a
part
icula
r D
NA
segm
ent
Screening with antibodies is used when cloned gene is
expressed and antibodies recognizing the encoded
protein are available.
(4). Amplified by polymerase chain reaction(PCR) method.
DNA polymerase DNA
template
primerprimer
Amplified
DNA
fragment
108
(5) Other methods
yeast one-hybrid
yeast two-hybrid
Yeast two-hybrid analysis
Cut
Cutting vector DNA and target DNA at precise
locations by proper restriction enzymes.
Choice of the restriction enzymes:
The target gene has two sites for one enzyme
in its two flanks.
The vector has one or two sites for the
enzyme at proper location or MCS
Using the
same one
enzyme to
cut target
gene and
vector
respectively
113
Choice of the restriction enzymes:
The target gene has two sites for
two different enzymes in its two
flanks respectively.
The vector has the same two sites
for the two enzymes in MCS
Using the
same two
enzymes to cut
the target
gene and
vector DNA
respectively
xxxGAATTCxxxxxxxxxxxAAGCTTxxx
xxxCTTAAGxxxxxxxxxxxTTCGAAxxx
EcoRI Hind III
AATTCxxxxxxxxxxxA
GxxxxxxxxxxxTTCGA
EcoRI
HindIII
G AGCTT
CTTAA A
Choice of the restriction enzymes:
The target gene has more than two
sites for one enzyme not only in the
two flanks but also within the gene.
The vector has the same site for the
enzyme in MCS
Using the
same one
enzyme to cut
the vector and
to digest the
target DNA
partiallyPstI PstI PstI
marker
sampleExcise the
interest band
and purify it
Ligate the gene
with the vector
that is cut by PstI
- +
Choice of the restriction enzymes:
Sometimes in the target gene there are no suitable
sites that also exist in the vector for some restriction
enzymes. How to deal with this problem.
Using the different enzymes that are suitable to cut out
the target gene from big DNA molecule
modifying the ends with some enzyme to form blunt
ends adding a linker with the site that also
exist in the vector to the modified blunt ends
best choice: PCR
Joining the target DNA
to the vector DNA
covalently using DNA
ligase to form a
recombinant DNA
Ligation
There are 3 kinds of ends ligated:
1. Ligation between compatible sticky ends
2. Ligation between blunt ends
3. Ligation between sticky and blunt ends
Ligase
AATTCxxxxxxA
GxxxxxxTTCGAG AGCTT
CTTAA A
GAATTCxxxxxxAAGCTT
CTTAAGxxxxxxTTCGAA
ligase
*Using the same one enzyme to cut
*Ligated easily and conveniently
*Self-reconnection of vector
*Inserted in two directions
Sticky-end ligation
*Using the same two enzymes to cut
*Ligated easily and conveniently
*no self-reconnection of vector
*Inserted in one direction
EcoRI HindIII
EcoRI HindIIIEcoRI EcoRI
Ligation between sticky ends with synthetic linker
GAATTCGG
CTTAAGCC
GGGAATTC
CCCTTAAG
EcoRI
Cut with RE
Ligase + linker
*Cut target DNA using proper RE
*Ligating blunt-end with a linker
*Cut the vector and DNA with a linker
using the same restriction enzyme and
ligate them.
Ligation between homopolymer tails
CCCCCC
CCCCCC
CCCCCC
GGGGGG
GGGGGG
CCCCCC
GGGGGG
GGGGGG
*To cut the DNA using the same
or different restriction enzymes
that produce 3’ sticky end.
*To add a homopolymer tail to
3’end using terminal transferase
dCTP terminal transferase
*Increasing ligation efficiency
*Decreasing self-reconnection
*2-direction insertion
*no easy way of retrieving the
insert
Generate restriction
site by PCR
123
引物设计时酶切位点的保护碱基表
124
The Taq polymerase has a nontemplate-dependent
terminal transferase activity that adds a single
deoxyadenosine (A) to the 3'-end of the PCR products
125
TOPO cloning is a molecular biology technique in which
DNA fragments amplified by Taq polymerase are cloned into
specific vectors without the requirement for DNA ligases.
Blunt-end ligation
Recombinant
DNA*to cut the DNA using
the same or different
enzymes that produce
blunt ends or sticky
ends that are trimed
to blunt ends by
exonuclease.
*more difficult ligation
*2-direction insertion
*self-reconnection
*no easy way of
retrieving the insert
T4 DNA ligase
vector
Ligation between
sticky and blunt ends
*Introduction of recombinant DNA into
recipient cells (host cells) is called
transformation
*Introduction of foreign DNA changes
(transforms) properties of the organism (host
cells).
Transformation: recombinant
plasmids into E.coli
*Special treatment makes cells competent -
capable of accepting foreign DNA.
*Usually, these treatments make cell membrane
more permeable for a DNA molecule.
0-4℃
Recombinant DNA + 42 ℃
The treatment of host cells:
1. Low-osmosis plus heat shock
E.coli + 0.1M CaCl2
competent (E.coli) cells
E.coli containing recombinant DNA
Low-osmosis makes cell expanding
Heat makes cell more expanding and
membrane more permeable
2. Electroporation makes holes of cell membrane with high voltage
calcium phosphate transfection
DEAE dextran-mediated transfection
Liposome/ Cationic polymer transfection
electroporation
microinjection
Transfection: recombinant DNA into
eukaryotes
134
图:倒置荧光显微镜观察pEGFP-
N1在Hela细胞中的表达效果
装置:电穿孔仪
A:普通电转杯B:装有镀金电极的Neon移液器吸头
Neon电转染系统
Microinjection system
DNA microinjection
137
Infection:
recombinant
viral into
eukaryotes
When competent cells are mixed with DNA, some
cells (actually, very few) become transformed:
they acquire recombinant vector DNA.
Pseudo-positive positive
Selection or screening
1. Selection according to the genetic markers
Antibiotic resistance: ampr , tetr , neor
Insertional inactivation
Marker rescue: β-galactosidase
Packaging properties of bacteriophage
140
2. Sequence specificity selction
Restriction map
PCR
Nucleic acid hybridization
DNA sequencing
3. Protein detection
All cells in
the clone are
genetically
identical
In each colony all cells
are descendants of one
transformed cell.
After transformation, cells are plated onto agar medium that
contains selective antibiotic: only transformed cells, that
acquire antibiotic resistance gene on the vector plasmid, will
survive and form colonies. All the untransformed cells will die.
1. Selection according to the genetic markers
antibiotic selection
Plated the cells on the
medium with amp or tet,
only transformed cells can
survive to become clones
Selection according to antibiotic resistance
EcoRI EcoRI
Insertional inactivation
The colonies surviving
only in tet, not in amp
are positive clones
phenotype: AmpsTetr
145
146
Marker rescue: Blue /white selection
148
BamHI
149
IPTG
150
Packaging
properties of
bacteriophage
RE map identification
2. Sequence specificity selction
155
*Incubating and amplifying the bacterial
colonies in proper liquid medium.
*Preparation and purification of plasmid
DNA from the bacteria.
*Cuting the plasmid DNA with the proper
restriction enzymes.
*separating the cut fragments with
electrophoresis.
M:DNA marker
1:purified PCR product of target DNA (2522bp)
2:recombinant plasmid/ Kpn Ⅰ+XhoⅠ(4.8kb+2.5kb)
3:empty plasmid/ Kpn Ⅰ+XhoⅠ (4.8kb)
4: recombinant plasmid /XhoⅠ (7.3kb)
separating the cut fragments with electrophoresis
157
BamHI BamHI
1000bpPst I
200bp 800bp
A BC
Identification of bidirectional
insertion by enzyme cut
BamHI2686bp+1000bp—positive
2686bp—pseudopositive
PstI
BA—200bp + 3486bp
AB—800bp+ 2886bp
4000
3000
2000
1000
200
PCR method
1.Negative control2.Positive control3.Recombinant plasmid4.DNA molecular marker
以提取的重组质粒为模板,利用序列特异性引物扩增插入片段,电泳鉴定正确后,送测序。
Use of hybridization to identify a
clone with a particular DNA
segment. The radioactive DNA
probe hybridizes to
complementary DNA and is
revealed by autoradiography.
Once the labeled colonies have
been identified, the orresponding
colonies on the original agar
plate can be used as a source of
cloned DNA for further study.
In situ hybridization
DNA sequecing
163
3.Protein detection
164
Separation: A specific gene or DNA
fragment and vector DNA that you
selected are separated from their
originals
Cut: Vector DNA and target DNA
are cut at precise locations by proper
restriction enzymes.
Ligation: The target DNA is linked
to the vector DNA covalently by
DNA ligase to form a recombinant
DNA.
Transformation: The recombinant
DNA is introduced into a host cell
that can provide the enzymatic
machinery for DNA replication.
Selection or screening: the host cells
that contain the recombinant DNA
are identified by using some methods
165
Step 6. Gene expression---the production of useful proteins/RNAs
Expression Systems
1. Need a portion of the transcript cDNA or
the gene that encodes the target protein
2. Need a DNA vector (this is usually a
plasmid carrying the cDNA or gene) to
direct transcription
3. Need cellular components that translate,
transcribe, and carry out any post-
translational modifications
Prokaryotic expression system
The bacteria expression system yielded a
high abundance of protein, but the final
protein was improperly folded and thus non-
functional.
Expression vectors of prokaryote should :
1.selection markers ;
2.power promotor;
3. Regulation sequences
4.Polylinker cloning sites
167
Eukaryotic Expression system
Yeast, insect , mammalian cells
Maintains at a high copy number(neo/G418)
High transcription efficiency (hCMV promoter)
High translation efficiency (5'-UTR)
Usually shuttle vectors
168