Cloning Strategies (cDNA LIB., Const & Screening)

Cloning strategies
Principles of Gene Manipulation by liuzengran

Hebei University of Economics and Business
Demands
should grasp the basic procedure of

DNA cloning and the techniques
involved in DNA cloning
Need to master the strategy to clone
a gene and the related methods.
Class-periods 8h

Main contents
6.1 Introduction
6.2. Cloning genomic DNA
6.3. cDNA cloning
6.4. Screening strategies

6.1 Introduction
Any cloning procedure has four essential parts:
(i) A method for generating the DNA fragment
for cloning; 基因片断获得
(ii) A reaction that inserts that fragment into
the chosen cloning vector; 重组质粒获得
(iii) A means for introducing that recombinant
vector into a host cell where it is replicated;
受体转化
(iv) A method for selecting recipient cells that
have acquired the recombinant 转化子筛选
Generalized overview of
cloning strategies

TA Cloning
exploits the terminal transferase activity of
some DNA polymerases.
This enzyme adds a single, 3'-A overhang to
each end of the PCR product.
The PCR products with dA overhang, are
mixed with this vector with single 3'-T
overhangs in high proportion.
The complementary overhangs of "T" vector
and PCR product will be ligated under the action
of T4 DNA ligase.

DNA Screening strategy
question
what should be done， when the DNA
donor source is very complex?
How
to rapidly sift through large numbers
of unwanted sequences
to identify the particular target?

two major strategies for isolating sequence
The first strategy
to divide the source DNA into manageable
fragments and clone everyone.
All the collection of clones is known as

a library， representative of the entire
starting population.
to identify the interest clone by screen the
library.
using a procedure to discriminate
between the desired clone and all
the others.
The second strategy
to selectively amplify the target sequence

directly from the source DNA, by polymerase
chain reaction (PCR),
clone this individual fragment.

Compare the two strategies
the library approach the PCR approach
the entire DNA the specific DNA

fragments are fragments are generated,
generated initially the screening step is not
 cloned need
indiscriminately only selected fragments
carry out screening are actually cloned

6.2. Cloning genomic DNA
6.2.1 Genomic DNA libraries
genomic library constructed
1 byλcloning vectors
2 byλreplacement vectors
3 by high-capacity vectors
6.2.2. PCR as an alternative to genomic DNA

cloning
6.2.2.1. Long PCR
6.2.2.2. Fragment libraries

6.2.1 Genomic DNA libraries
6.2.1.1 Constructing representative genomic
libraries in λcloning vectors
How to clone a single-copy gene from the

human genome?
methods
digest total human DNA with a
restriction endonuclease
insert the fragments into a
suitable phage-λ vector
isolate the desired clone.

How many recombinants have to be screened ?
Assuming
Human haploid(单倍体） genome (2.8 × 10 6 kb)
EcoRI Fragments of about 4 kb
over 7 × 105 independent recombinants must

be prepared and screened

How can appropriate size of random
fragments be produced?
Random breakage by mechanical shearing
the average fragment size can

be controlled,
insertion reaction requires
additional steps (to produce the
stick end)
restriction endonucleases.

卡隆载体
random
Maniatis’
strategy for
producing a
representative
gene library.

in this approach, There are two problems.
the gene the obtained gene

may be cut may be larger than the
internally vector can accept.
it is not obtained the appropriate

as a single gene would not be
fragment. cloned

Solution
by cloning random DNA fragments of a large

size (for λ replacement vectors, ～ 20 kb)
fewer clones are required

for a complete or nearly
complete library.

6.2.1.2 Development of λ replacement
vectors for genomic library construction
Due to the convenience and efficiency
the λEMBL series of vectors have

been widely used for genomic
library construction .

Creation of a genomicPrinciples
DNA of Gene Manipulation by liuzengran
library using the phage-λ vector EMBL3A.
6.2.1.3 Genomic libraries in high-capacity vectors
a number of higher capacity cosmids,

cloning vectors are available for (BACs),
the construction of genomic (PACs)
libraries. (YACs)
advantage
the number of recombinants
the insert needed to be screened is lower,
size is much large genes are more likely to
larger than be contained within a single
for λ clone
replacement fewer steps are needed for a
vectors. chromosome walk
6.2.2. PCR as an alternative to genomic DNA cloning
The PCR is a powerful technique for amplifying
specific DNA sequences from complex sources.
limitation 仅适于小片段
standard PCR conditions are
suitable only for the
amplification of short products.
The maximum product

size is about 5 kb,
the typical size is 1–2 kb.

6.2.2.1. Long PCR
6.2.2.2. Construct fragment libraries by PCR

6.2.2.1. Long PCR－改变反应条件/双聚合酶
Modify the reaction

Prevent the template
conditions
from damage
( lower the reaction
temperature and
improve polymerase
increase the pH)
processivity
+
Proof reading enzyme，
two DNA polymerases
Taq polymerase.
improve the performance of PCR

of long templates
As no proofreading
PCR with Taq polymerase activity
mismatched bases are incorporated

into growing DNA strands.
proofreading enzyme
removes mismatched bases
Result
possible to amplify DNA fragments （up to 22
kb ），directly from human genomic DNA.

6.2.2.2. Construct fragment libraries by PCR
the PCR can be used to generate

libraries, i.e. by amplifying a
representative collection of random
genomic fragments.
Construct libraries of small amounts of

starting material, ( e.g. single cells or
fixed tissue)

How to construct a genomic library by PCR ?
2 methods used
digest genomic DNA using random
with restriction enzymes, primers
ligate linkers to the ends Select for

of DNA fragments, （to suitable PCR
provide annealing sites for products by
one specific type of size
primer ）.

6.3. cDNA cloning
6.3.1 Preparation of cDNA for library construction
6.3.1.1 The cDNA synthesis reaction

6.3.1.2 Development of cDNA cloning strategies
6.3.2 Full-length cDNA cloning

6.3.3 PCR as an alternative to cDNA cloning

6.3.1 Preparation of cDNA for library construction
6.3.1.1 The cDNA synthesis reaction(RT-PCR)
involves three major steps:
(to synthesis a double-stranded cDNA)
first-strand DNA synthesis on the mRNA

template (with a reverse transcriptase) ;
removal of the RNA template;
Second strand DNA synthesis using the
first DNA strand as a template, (with a DNA-
dependent DNA polymerase).

6.3.1.2 Development of cDNA cloning strategies

1 An early cDNA cloning strategy
the first cDNA strand has the

tendency to transiently fold
back on itself.
a hairpin loop

A serious disadvantage of the hairpin method
is that cleavage with S1 nuclease results in
the loss of a certain amount of sequence at
the 5′ end of the clone.
2 Improved method
a full-length cDNA
lower efficiency

For cDNA expression libraries,
it is advantageous
if the cDNA can be inserted into the vector
in the correct orientation.- (定向克隆)

3 adding a synthetic linker to the double-stranded cDNA
before the hairpin loop is cleaved

4 using an
oligo-dT
primer
containing a
linker
sequence
direction
cloning
achieved

5 using primers that are already linked to a plasmid

The methods to achieve direction cloning
(定向克隆) for expression ---3 methods
adding a linker to the double-stranded cDNA
before the hairpin loop is cleaved
在发夹结构切除前添加linker
using an oligo-dT primer containing a linker

sequence when the second strand is primed.
用含 oligo-dT 的primer合成第二链
using primers for cDNA synthesis that are

already linked to a plasmid
用已经连在质粒上的引物合成cDNA
7 The Gubler–Hoffman method---widely used
a simple and general method for
non-directional cDNA cloning.
nick-translation
high efficient

6.3.2 Full-length cDNA cloning
6.3.2.1 Limitations of conventional clone strategies
(of cDNA libraries) 传统克隆策略缺陷
Two drawbacks
1 trigger a 3′-end bias
oligo-dT be addressed through

primers are the use of random
used to initiate oligonucleotide primers,
first-strand for both first- and
synthesis, second-strand cDNA
synthesis.
2 difficult to isolate full-length clones
(when the size of a cDNA increases)
partly due to deficiencies in the reverse-

transcriptase enzymes used for first-strand
cDNA synthesis.
Native enzymes have poor processivity

and intrinsic RNase activity,
leads to degradation of the

RNA template

6.3.2.2 Selection of 5 ′mRNA ends
Strategy
all eukaryotic mRNAs have a 5′end cap
Combining cap selection and

nuclease treatment
to select for full-length first-strand cDNAs
generate libraries
(highly enriched in full-length clones).

The capture method
an oligo-dT primer of full-length cDNA
cloning,
hybrid molecules
protected
digested digests single-
away stranded RNA
eukaryotic
initiation factor
using the eIF-4E to select

mRNAs with caps

An alternative method, oligo-capping(寡聚加帽法）
full-length broken and degraded

molecules be ligated molecules cannot be
to a specific ligated to a specific
oligonucleotide oligonucleotide
An oligo-capped population of
full-length mRNAs resulted

oligo-capping
alkaline phosphatase 碱性磷酸酶 removes phosphate
acid pyrophosphatase 酸性焦磷酸酶 removes the cap
The basis of the

method is that
RNA is treated
with alkaline
phosphatase and
an oligo-capped population
of full-length mRNAs. acid
pyrophosphatase.
using the oligo-dT

primer and a
primer annealing to
the oligonucleotide
cap. Hebei University of Economics and Business
6.3.3 PCR as an alternative to cDNA cloning
6.3.3.1 RT-PCR
amplify RNA sequences in

cDNA form.

Features of Reverse transcription
Using a specific 3′ primer to generate

the first cDNA strand,
initiate the PCR amplification by adding a
5′primer to the reaction mix.
used the total RNA as the starting
material.
用专一性 3′引物合成cDNA第一链。
向混合物中直接添加5′引物起始反应
用总RNA 作为模板.

RT-PCR can be used to provide the cDNA for
library construction, when the source is
unsuitable for conventional approaches.
universal primers
amplification of all mRNAs
subcloned into suitable vectors.
cDNA library

Disadvantages of the PCR-based approaches(3)
depend upon specific primers
the DNA polymerases are more error-prone
library may contain many mutations

Competition among templates, and a bias
towards shorter cDNAs.
certain amount of distortion may produced
contaminating genomic sequences.
be amplified and false results resulting

disadvantage of building cDNA library
screening a cDNA library is laborious,
may not yield a cDNA clone with a full-

length coding region,
the sought-after cDNA may be very rare

6.4. Screening strategies 利用序列/表达产物筛选
To identify a specific clone from a DNA

library, two methods can be used.
exploiting of the sequence of the clone

exploiting of the structure/function of its
expressed product
In the two processes
the probe or primers be used

attention
Screening the product of a clone

applies only to expression libraries,
the DNA fragment is

expressed to yield a
protein.

6.4.1. Sequence-dependent screening
6.4.1.1. Screening by hybridization
6.4.1.2. Screening by PCR
6.4.2. Screening expression libraries

6.4.2.1. Immunological screening
6.4.2.2 Screening with alternative ligands
6.4.2.3. Functional cloning
6.4.2.4. Screening by functional complementation
6.4.2.5. Screening by ‘gain of function’

6.4.1. Sequence-dependent screening
6.4.1.1. Screening by hybridization
Nucleic acid hybridization is the most

commonly used method of library screening
feature
rapid,
applied to very large numbers of clones
be used to identify clones that are not full-
length (cannot be expressed),(in the cDNA
libraries)

replica
The filter is applied to the upper surface

of agar plates, making direct contact

菌落杂交 hybridization
Colony hybridization in situ with
radioactive
RNA probes
sodium hydroxide

Control plate
plaque lift
Plaque contain phage 嗜菌斑转移
particles and unpackaged
recombinant DNA.

probe
to allow for the presence

of mismatches
6.4.1.2. Screening by PCR
if there is sufficient information about its
sequence
make suitable primers
with gene-specific
to identify any primers
clone by PCR SO
a specific clone can
be isolated by PCR
method.

6.4.2. Screening expression libraries
using expression vectors
if
the DNA sequence of the target clone is
unknown
no available strategy to design a suitable

probe or primers.
Relay on
expression libraries

6.4.2.2 South-western and north-western
screening
6.4.2.4. Screening by functional
complementation

one of the most versatile
expression cloning strategies,
the prerequisite is the proper

antibody
It does not matter whether the
protein be functional.
The recognition target is generally
an epitope(抗原决定基)

folds into a particular three-
Epitopes dimensional conformation on
the surface of the protein.
a short sequence of amino acids

properties
can fold independently of the rest of the
protein
can fold when:
the polypeptide chain is incomplete
expressed as a fusion with another protein

The steps of the first immunological
screening techniques
1. Transformed cells were inoculated on 接种培养

Petri dishes and allowed to form colonies. 形成菌落
2. The colonies were lysed to release the 阳性菌落

antigen from positive clones, 裂解
3. A sheet of polyvinyl coated with the 将涂有合

appropriate antibody was put onto the 适抗体的
聚乙烯膜
surface of the plate,
置于培养
antigen–antibody complexes formed. 皿上

4. The sheet was
removed and exposed
to 125I-labelled IgG
specific to a different
determinant（决定因子）
on the surface of the
antigen
5. The sheet was then

washed and exposed
to X-ray film.
Antigen–antibody
complex
Pay attention Epitope
This ‘sandwich’ technique 三明治方法要求

need a protein, having two 蛋白质有两个不
different determinants, 同的决定因子，
可以被不同抗体
识别
be recognized by
different antibodies

Example
Immunological screening with λ cDNA
libraries using the expression vector λgt11
or
β-galactosidase

6.4.2.2. South-western and north-western screening
South-western
This approach has been used for the
screening and isolation of clones (protein)
That express sequence-specific
Method DNA-binding proteins.
incubating the membranes (fixed with
expression protein) with a radio labelled double
stranded DNA oligonucleotide probe,
containing the recognition sequence
for the target DNA-binding protein.
north-western screening
using a single-stranded RNA

probe to isolate sequence-
specific RNA-binding proteins

functional positional
cloning cloning
screening methods depend on the biological
activity of the protein..
be ignorant of the position of the gene in the

genome
requires no prior knowledge:
 of the nucleotide sequence of the clone
 the amino acid sequence of its product.

steps
the expressed protein is functional
to screen an
expression library
the corresponding clone be identified.

6.4.2.4. Screening by functional complementation
Functional complementation is a very

powerful method of expression cloning
Functional complementation is the process
a particular DNA sequence compensates

for a missing function in a mutant cell
the wild-type phenotype restored.

under particular growth conditions
mutant of interest gene are non-viable
Wild-type are viable
cells carrying the clone of interest

can be positively selected
the corresponding clones be isolated.

Functional
complementation
used in
transgenic animals.
microinject
Functional complementation
in transgenic mice to isolate
the Shaker-2 gene.Hebei University of Economics and Business
problem
can be used only if an appropriate
mutant expression host is available.
loss of function in the host may be

fully or partially compensated by
one or more other genes.
Screening by ‘gain of function’

Clones confer a gain of function of host cell.
it may be possible to identify clones
is
gain of function is/or not a Is not
selectable phenotype
Allows cells the phenotype conferred

containing the by the clone of interest is
corresponding clone not selectable
to be positively The gained phenotype
selected. causes a visible
change in phenotype
be detected

Cloning Strategies (cDNA LIB., Const & Screening)

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Cloning strategies

Principles of Gene Manipulation by liuzengran

should grasp the basic procedure of

Principles of Gene Manipulation by liuzengran

6.2. Cloning genomic DNA

6.3. cDNA cloning

6.4. Screening strategies

Principles of Gene Manipulation by liuzengran

Principles of Gene Manipulation by liuzengran

Principles of Gene Manipulation by liuzengran

Principles of Gene Manipulation by liuzengran

All the collection of clones is known as

to selectively amplify the target sequence

clone this individual fragment.

Principles of Gene Manipulation by liuzengran

the library approach the PCR approach

the entire DNA the specific DNA

Principles of Gene Manipulation by liuzengran

6.2.2. PCR as an alternative to genomic DNA

Principles of Gene Manipulation by liuzengran

How to clone a single-copy gene from the

Principles of Gene Manipulation by liuzengran

Human haploid(单倍体） genome (2.8 × 10 6 kb)

EcoRI Fragments of about 4 kb

over 7 × 105 independent recombinants must

Principles of Gene Manipulation by liuzengran

the average fragment size can

Principles of Gene Manipulation by liuzengran

Principles of Gene Manipulation by liuzengran

the gene the obtained gene

it is not obtained the appropriate

Principles of Gene Manipulation by liuzengran

by cloning random DNA fragments of a large

fewer clones are required

Principles of Gene Manipulation by liuzengran

Due to the convenience and efficiency

the λEMBL series of vectors have

Principles of Gene Manipulation by liuzengran

a number of higher capacity cosmids,

The maximum product

Principles of Gene Manipulation by liuzengran

6.2.2.2. Construct fragment libraries by PCR

Principles of Gene Manipulation by liuzengran

Modify the reaction

improve the performance of PCR

mismatched bases are incorporated

Principles of Gene Manipulation by liuzengran

the PCR can be used to generate

Construct libraries of small amounts of

Principles of Gene Manipulation by liuzengran

ligate linkers to the ends Select for

Principles of Gene Manipulation by liuzengran

6.3.1 Preparation of cDNA for library construction

6.3.1.1 The cDNA synthesis reaction

6.3.2 Full-length cDNA cloning

Principles of Gene Manipulation by liuzengran

first-strand DNA synthesis on the mRNA

Principles of Gene Manipulation by liuzengran

Principles of Gene Manipulation by liuzengran

the first cDNA strand has the

Principles of Gene Manipulation by liuzengran

Principles of Gene Manipulation by liuzengran

Principles of Gene Manipulation by liuzengran