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Recombinant DNA
Technology
Dr. Hui LI
Office : S408
Tel: 26538722
Process of cloning
• Isolation of target gene
• Selection and construction of
vectors
• Ligation of target DNA and vector
• Transformation of target gene into
receptor cell
• Screening for recombinant plasmids
• Expressing a cloned gene
Process of DNA cloning
§1. Isolation of target gene
1. Chemical synthesis
only for simple polypeptide chain
whose primary structure is clear.
2. Obtaining from genomic DNA library
3. Obtaining from cDNA library
4. polymerase chain reaction (PCR)
The genomic
DNA library is a
collection of the
comprehensive
DNA fragments
representing the
entire genome of
a species.
The cDNA library
represents the
population of
mRNAs, it only
contains the exons
of protein’s
structural genes.
mRNA
Reverse transcripase
cDNA
replication
dscDNA
vector
recombinate DNA
E. coli
recombinate DNA in E.coli
§1.1 Chemical synthesis of a gene
 Oligonucleotide synthesis is the chemical synthesis of relatively short
fragments of nucleic acids with defined chemical structure (sequence).
 The technique is extremely useful in current laboratory practice because it
provides a rapid and inexpensive access to custom-made oligonucleotides of
the desired sequence.
 Whereas enzymes synthesize DNA and RNA in a 5' to 3' direction, chemical
oligonucleotide synthesis is carried out in the opposite, 3' to 5' direction.
 Currently, the process is implemented as solid-phase synthesis using
phosphoramidite method (固相亚磷酸三酯法). The occurrence of side reactions
sets practical limits for the length of synthetic oligonucleotides (up to about
200 nucleotide residues) because the number of errors accumulates with the
length of the oligonucleotide being synthesized.
 Products are often isolated by HPLC to obtain the desired oligonucleotides
in high purity.
Each nucleotide addition consists of
four chemical reactions: deblocking
(detritylation), coupling, oxidation, and
capping.
 During deblocking, DMT is removed
from the growing oligonucleotide
resulting in a free 5’ hydroxyl group.
 During
coupling,
an
activated
nucleoside phosphoramidite is joined to
the newly formed 5’ hydroxyl.
 During
oxidation,
the
unstable
tricoordinated phosphate triester
linkage formed is converted to a
protected tetracoordinated phosphate
triester.
Oligonucleotide synthesis cycle
 During capping, oligonucleotides that
fail to join to an activated nucleoside
are permanently blocked to prevent
their further use in subsequent steps.
Figure adapted from (Caruthers et al. 1987).
Oligonucleotide synthesis is carried out by a stepwise addition of nucleotide
residues to the 5'-terminus of the growing chain until the desired sequence is
assembled. Each addition is referred to as a synthetic cycle and consists of
four chemical reactions:
Step 1 - De-blocking (detritylation): The DMT group （二甲氧基三苯甲基）is
removed with a solution of an acid, such as TCA (三氯乙酸)，in an inert
solvent and washed out, resulting in a free 5' hydroxyl group on the first
base;
Step 2 - Coupling: A nucleoside phosphoramidite (or a mixture of several
phosphoramidites 亚磷酰胺 ) is activated by an acidic azoles catalyst,
tetrazole (四唑), 2-ethylthiotetrazole, 2-bezylthiotetrazole, 4,5dicyanoimidazole, or a number of similar compounds. This mixture is
brought in contact with the starting solid support (first coupling) or
oligonucleotide precursor (following couplings) whose 5‘-hydroxy group
reacts with the activated phosphoramidite moiety of the incoming
nucleoside phosphoramidite to form a phosphite triester linkage（亚磷酸三
酯键）. This reaction is very rapid and requires, on small scale, about 20 s
for its completion. The phosphoramidite coupling is also highly sensitive
to the presence of water and is commonly carried out in anhydrous
acetonitrile（ 乙腈）. Unbound reagents and by-products are removed by
washing
Step 3 - Capping: After the completion of the coupling reaction, a small
percentage of the solid support-bound 5'-OH groups (0.1 to 1%) remains
unreacted and needs to be permanently blocked from further chain
elongation to prevent the formation of oligonucleotides with an internal
base deletion commonly referred to as (n-1) shortmers. This is done by
acetylation (乙酰化作用)of the unreacted 5'-hydroxy groups using a mixture
of acetic anhydride and 1-methylimidazole as a catalyst. Excess reagents are
removed by washing.
Step 4 - Oxidation: The newly formed tricoordinated phosphite triester
linkage （亚磷酸三酯键） is not natural and is of limited stability under the
conditions of oligonucleotide synthesis. The treatment of the support-bound
material with iodine and water in the presence of a weak base (pyridine,
lutidine, or collidine) oxidizes the phosphite triester into a tetracoordinated
phosphate triester (磷酸三酯键), a protected precursor of the naturally
occurring phosphate diester internucleosidic linkage.
Synthetic cycle for preparation of oligonucleotides by phosphoramidite method
(Left) Polymerase Cycling
Assembly (PCA) synthesis
procedure.
Oligonucleotides with
complementary regions are
pooled and repeated
annealing and polymerase
extension cycles assembly
and amplify a full-length
product.
(Right) Ligase Cycling
Assembly (LCA) synthesis
procedure.
Using a thermostable ligase,
repeated annealing and
ligation cycles join
oligonucleotides into
increasingly larger strands.
A final PCR step is often
employed to amplify the
full-length target from the
incomplete products.
Overview of PCA and LCA.
Normally, at the end of each procedure, 30-50
oligonucleotides are synthesized. They are most
commonly used as antisense oligonucleotides,
small interfering RNA, primers for DNA
sequencing and amplification, probes for
detecting complementary DNA or RNA via
molecular hybridization, tools for the targeted
introduction of mutations and restriction sites,
and for the synthesis of artificial genes.
1.2 Polymerase Chain Reaction
The polymerase chain reaction (PCR) is
a rapid and versatile in vitro method for
amplifying DNA.
PCR reaction system
•
•
•
•
•
DNA template
A pair of primers
DNA polymerase (ex. Taq)
dNTPs
Mg2+-containing buffer
Procedures of PCR
• Denaturing: the template DNA is
denatured to become ssDNA from
dsDNA by heating.
• Annealing: this step allows the
hybridization of the primers with
target DNA.
• Extension: this process is the DNA
synthesis step.
ing
The first three
cycles of PCR
§1.3 Isolating a gene from gene library
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
Gene library
(made from genomic DNA)
cDNA libraries
(made from cDNA- copy of mRNA)
§1.3.1 Genomic DNA libraries
1. Purify genomic DNA： prokaryotes or
eukaryotes;
2. Fragment this DNA : physical shearing
and restriction enzyme digestion;
3. Clone the fragments into vectors;
4. Transfer the constructed vectors into
recipient cells;
5. Culture and amplify the clones;
6. Screen the clone of target.
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 :
Eukaryotes :prepare cell nuclei
remove protein, lipids and other unwanted macromolecules by protease digestion and phase extraction.
Prokaryotes :extracted DNA directly from cells
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.
Genomic libraries
Selection of restriction enzyme
1. Ends produced (sticky or blunt) &
The cleaved ends of the vector to be cloned
Sau3A: 5’-/GATC-3’, less selectivity
BamH1: 5’-G/GATCC
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.
Genomic libraries
Vectors
According to genome’s size,we can select a
proper vector to construct a library .
Vectors
Plasmid
insert (kb)
5
λ phage
cosmid
23
45
YAC
1000
The most commonly chosen genomic cloning vectors
are λ replacement vectors which must be digested with
restriction enzymes o produce the two λ end fragment
or λ arms between which the genomic DNA will be
digested
Genomic libraries
λ phage vector in cloning
Long (left)
arm
cos
short (right)
arm
Exogenous DNA
(~20-23 kb)
Long (left)
arm
cos
short (right)
arm
cos
cos
Exogenous DNA
(~20-23 kb)
0.preparation of
arm and genomic
inserts
1. Ligation
λ replacement
vector cloning
2. Packing with a
mixture of the
phage coat proteins
and phage DNAprocessing enzymes
3. Infection and
formation of
plaques
Library constructed
§1.3.2 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
cDNA libraries
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.
cDNA libraries
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
mRNA isolation
• Most eukaryotic mRNAs are polyadenylated at
their 3’ ends
5’ cap
AAAAAAAAAAn
• oligo (dT) can be bound to the poly(A) tail
and used to recover the mRNA.
I 2 cDNA libraries
cDNA libraries
Three methods to isolate mRNA.
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 mRNAribosome complexes on sucrose gradients
cDNA libraries
Check the mRNA integrity
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
cDNA libraries
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)
cDNA libraries
Synthesis of cDNA :
First stand synthesis: materials as
reverse transcriptase ,primer( oligo(dT) or
hexanucleotides) and dNTPs
(Fig 1)
Second strand synthesis: best way of
making full-length cDNA is to employs a
ribonuclease ( RNase H ) which recognises
the RNA component of a DNA:RNA hybrid
and cleaves the RNA at a number of nonspecific sites .(Fig2)
Fig.1 First strand synthesis
Fig.2 Second strand synthesis
cDNA libraries
Treatment of cDNA ends
Blunt end 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)
Tailing with terminal transferase or
using adaptor molecules
Restriction enzyme digestion (E.coRI )
cDNA libraries
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)
Screening procedures
1.2.3 Screening procedures
1 Screening
2 Colony and plaque hybridization
3 Expression screening
4 Hybrid arrest and release
5 Chromosome walking (repeat screening)
Screening procedures
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.
Screening procedures
Screening libraries
Searching the genes of interest in a DNA library
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
Screening procedures
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
(Alkali treatment)
Containing Nucleic acid probe)
X-ray
film(radioactively
labeled )
Wash to remove unhybridization probe and visualize
Line up the hybridizated region or
repeated hybridization
antibody or
enzyme
(modified
nucleotide
labeled
Screening procedures
Transfer to nitrocellulose
or nylon membrane
Keep master
plate
Select positive
from master plate
Probe with 32p-labled DNA
complementary to
gene of interest
Expose to film
Screening by plaque hybridization
Denature DNA(NaOH)
Bake onto membrane
Screening procedures
3 Expression screening
Identify the protein product of an
interested gene
1. Protein activity
2. Western blotting using a specific
antibody
Screening procedures
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).
Screening procedures
Expression screening (2)
Antibodies can be used to screen the
expression library.
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
Screening procedures
4 Hybrid arrest and screen
Individual cDNA clones or pools of clones can
be used to hybridize to mRNA preparation
Hybrid arrest :translate the mRNA population
directly, and the inhibition of translation of
some products detected.
Hybrid release translation : purify the
hybrids and the hybridized mRNAs released
from them and translated, it identifies the
protein encoded by the cDNA clone
Screening procedures
5 Chromosome walking
Definition: To clone the desired gene by
repeated isolating adjacent
genomic clones from the library.
to obtain overlapping genomic clones
that represent progressively longer
parts of a particular chromosome .
Screening procedures
Process:
1. Prepare a probe from the end insert .
2.The probe are used to re-screen the library
by colony or plaque hybridization
3.Analyzed the new isolate clones and posited
them relative to the starting clone.
some will be overlapping.
4. Repeated the whole process using a probe
from the distal end of the second clone.
Vector arm
}
}
Restriction
Genomic clone insert
Vector arm
Prepare probe from
ends of insert
Re-screen genomic
library
Restriction map new
genomic clones
}
}
Prepare new probes from distal ends of least overlapping insert.
Re-screen genomic library . Restriction map new genomic clones
Chromosome walking
Chromosome walking
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