Section I
Gene libraries and screening
I 1 Genomic libraries
I 2 cDNA Libraries
I 3 Screening Procedures
Section I: Gene libraries and screening
Yang Xu, College of Life Sciences
I 1 Genomic libraries
•
•
•
•
Gene libraries
Size of libraries
Genomic DNA
Vectors
Section I: Gene libraries and screening
Yang Xu, College of Life Sciences
Gene libraries
Gene library
Genomic
libraries
cDNA
libraries
Gene library: It is a collection of different DNA sequences from an
organism, each of which has been cloned into a vector for ease of
purification, storage and analysis.
(1) Genomic libraries: Gene libraries made from genomic DNA;
(2) cDNA libraries: If the DNA is a copy of an mRNA population,
that is cDNA, then the gene library is called a cDNA library.
Structure of representative gene libraries:
• Containing all the original sequences;
• Containing the necessary restriction sites;
• Containing a sufficient number of clones, otherwise some genes
will be missing.
Section I: Gene libraries and screening
Yang Xu, College of Life Sciences
Size of libraries
• Recombinant number: A gene library must contain a certain
number of recombinants, so as to have a high probability of
any particular sequence.
• The recombinant (transformants) number (N): can be
calculated if the genome size and the average size of the insert
in the vector are known. The formula is:
N = ln (1-P) / ln (1-f)
where P is the desired probability and f is the fraction of the
genome in one insert.
• For example:  desired probability is 0.99;  insert sizes is 20
kb and  the size for the E.coli is 4600 kb.
N = ln (1-0.99) / ln [1-(20/ 4,600)] = 1100
Why it is not 4600/20=230？
Section I: Gene libraries and screening
Yang Xu, College of Life Sciences
Genomic DNA
Making of a representative genomic library:
Genomic DNA must be  purified and  broken
randomly into fragments with correct size for
 cloning into the chosen vector.
Purification:
• Prokaryotes can be extracted by phase extraction;
• Eukaryotes  first preparing cell nuclei and
 then removing unwanted proteins and lipids
by protease digestion and phase extraction.
Breaking: There are physical shearing and restriction digestion.
• Physical shearing: Sonication will break the DNA into smaller
fragments.
• Partial digestion: to generate genomic DNA fragments of 15-25 kb
(a convenient size for  and cosmid vectors). Example: Sau3A
cleaves to produce a sticky end that is compatible with a vector
that has been cut with BamH1
Section I: Gene libraries and screening
Yang Xu, College of Life Sciences
Vectors for Genomic Libraries
Plasmids
 phage
10 kb
23kb
cosmid
45kb
YAC
BAC
1000 kb
350kb
Vectors: Plasmids,  phage, cosmid, YAC or BAC vectors can be
used to construct genomic libraries. The choice depends on the
genome size.
Segment size: The upper size limit of these vectors is about 10, 23,
45, 1000 and 350 kb respectively.
Section I: Gene libraries and screening
Yang Xu, College of Life Sciences
I 2 cDNA libraries
1. mRNA isolation, purification and fractionation
2. Synthesis of cDNA
3. Treatment of cDNA ends
4. Ligation to vector
Section I: Gene libraries and screening
Yang Xu, College of Life Sciences
mRNA isolation and purification
mRNA source for cDNA library making:
• They are not made from prokaryotic mRNA, since it is
very unstable;
• Making cDNA libraries from eukaryotic mRNA is very
useful because cDNAs have no intron and can be
expressed into proteins in E. coli.
mRNA isolation and purification:
• Total RNA: by extracting lysed cells with phenolchloroform;
• Isolating mRNA: Oligo(dT) can be bound to the poly (A)
tail of mRNA and used to recover the mRNA.
Section I: Gene libraries and screening
Yang Xu, College of Life Sciences
mRNA isolation and purification
• Isolating mRNA: Oligo(dT) can be bound to the poly (A)
tail of mRNA and used to recover the mRNA.
– Traditional method: This was done by passing a
preparation of total RNA down a column of oligo
(dT)-cellulose;
– New Method: To keep damage by nuclease to a
minimum, the more rapid procedure of adding
oligo(dT) linked to magnetic beads directly to a cell
lysate and “pulling out” the mRNA using a strong
magnet.
Section I: Gene libraries and screening
Yang Xu, College of Life Sciences
mRNA function check
Checking the integrity of an mRNA: Before using an mRNA
preparation for cDNA cloning, it is advisable to check that it is
not degraded. Main two methods are as below:
• Translation: See if the protein expression.
– Cell-free translation systems: such as wheat germ extract or
rabbit reticulocyte lysate;
– Microinject: to put the mRNA preparations into cells to
check whether their proteins are translated.
• Gel electrophoresis: It is also a common way.
– The mRNA preparation: usually produces a smear of
molecules from about 0.5 kb up to about 10 kb or more;
– The two largest rRNA: contaminate the mRNA preparations
because of their abundance and appear as clear bands within
the smear of mRNA.
Section I: Gene libraries and screening
Yang Xu, College of Life Sciences
Subtracted cDNA library
Subtracted cDNA library: to make a cDNA
library of all the mRNA sequences just in the
induced cells, one would prepare mRNA
from both induced and non-induced cells.
• ssDNA: are made from the non-induced cell
mRNA;
• mRNA: are extracted from the induced cells;
• [ssDNA/mRNA]: the complementary
sequences of both ssDNA and mRNA
hybridize each other to form duplexes.
• The rest mRNA: is newly induced mRNA,
which could then be isolated (e.g. using
magnetic beads) for constructing subtracted
cDNA library.
Section I: Gene libraries and screening
Yang Xu, College of Life Sciences
Synthesis of cDNA
mRNA
The first DNA strand making:
AAAAA-3’
5’
– A primer to extend the
RT trans. HO-TTTTTp-5’
mRNA template. This
4 dNTP
primer is usually oligo(dT);
mRNA
– All four dNTPs and reverse
AAAAA-3’
5’
transcriptase must be added;
3’
TTTTTp-5’
cDNA
– Adding the terminal
terminal transferase
transferase and dCTP;
mRNA
– Alkali for hydrolyzes
CCC-3’
AAAAA-3’
5’
TTTTTp-5’
RNA and purify the DNA;3’-CCCCCCCC 3’
cDNA
Alkali for RNA
The second DNA strand
5’-pGGGG-OH
making:
3’-CCCCCCCC
3’
TTTTTp-5’
cDNA
– Adding reverse transcriptase
(or Klenow polymerase)
5’-pGGGG
3’
-3’
and four dNTPs.
3’-CCCCCCCC
3’
Section I: Gene libraries and screening
cDNA
TTTTTp-5’
Yang Xu, College of Life Sciences
Treatment of cDNA ends
Solution: Blunt end ligation of large fragments is not efficient, usually, special
nucleic acid linkers are added to create sticky ends for cloning.
5’-pGGGG
3’
3’-CCCCCCCC
3’
-3’
TTTTTp-5’
1.
5’-pGGGG
3’
3’-CCC
3’
-3’
TTTTTp-5’
5’-pGGGG
3’
3’-CCCC
3’
-3’
TTTTTp-5’
2.
3.
5’-pGGGG
3’
HO-CCCAATTCGGGGGG
3’-CCCC
3’-GGCTTAAGCCCCCC
3’
CCGAATTCGG-3’
-3’
GGCTTAAGCC-OH
TTTTTp-5’
4.
5’-pGGGG
5’-pAATTCGGGGGG
3’
3’-CCCC
3’-GCCCCCC
3’
Section I: Gene libraries and screening
CCG-3’
-3’
GGCTTAAp-5’
TTTTTp-5’
Yang Xu, College of Life Sciences
Ligation to vector
Ligation to vector: Any vector
with an EcoRI site will be
suitable for cloning the cDNA.
• Dephosphorylate: It is usual to
dephosphorylate the vector
with the enzyme alkaline
phosphatase as this will
prevent the vector fragments
(ends) rejoining during ligation.
• Ligation: It is carried out using
T4 DNA ligase, and the
reeombmant molecules are
either packaged (see Topic H2)
or transformed (see Topic G2)
to create the cDNA library.
Section I: Gene libraries and screening
E
E
E S
E
G -OH
HO -AATTC
CTTAA -OH
HO -G
G -OH
P
CTTAA -OH
HO -AATTC
P
HO -G
Yang Xu, College of Life Sciences
I 3 Screening Procedure
•
•
•
•
Screening for libraries
Colony and plaque hybridization
Expression screening
Hybrid arrest and release
Section I: Gene libraries and screening
Yang Xu, College of Life Sciences
Screening for Libraries
Definition: The process of identifying one particular clone
containing the target gene from among the very large
number of others in the gene library is called the screening
for libraries.
Nucleic acid probe design: The sequence mainly comes from:
 Some knowledge from gene bank database or papers about
the target gene may be used for making a nucleic acid probe.
 If sufficient of the protein product is available to permit
determination of some ammo acid sequence, which can be
used to derive possible DNA sequences to make a mixture of
probes.
Making of the probes: Automated chemical synthesis and then
followed by PCR propagation.
Screening methods:
1. Hybridization with probe; 2. Expression screening;
3. Hybrid arrest and release translation; 4. Chromosome
walking.
Section I: Gene libraries and screening
Yang Xu, College of Life Sciences
Colony and plaque hybridization with probe-I
Definition: Hybridization with probe is an important method for
screening a single clone which contains  gene library. Although
 gene libraries produce plaques not colonies, after the initial
step, both hybridization ways are the same.
Transfer to nitrocellulose
or nylon membrane
Keep master plate
Select positive
from master plate
Denature DNA (NaOH)
Bake on to membrane
Probe with 32P-labeled
DNA complementary to
Gene of interest
Expose to film
Section I: Gene libraries and screening
Yang Xu, College of Life Sciences
Colony and plaque hybridization with probe-II
1. Transferring: Some of the DNA
in the plaque or colony can be
transferred to a nylon or
nitrocellulose membrane, after
which are placed on top of the
master plate.
2. Replica plating: Growing a colony replica on another dish directly
on the membrane surface.
3. Lysing: The bacteria on the membrane are lysed by soaking in
SDS and a protease, so that the colonies release DNA.
4. Denaturing and baking: The DNA on the membrane is then
denatured with alkali (NaOH) to produce single strands, which
are bonded to the membrane by baking or UV irradiation.
Section I: Gene libraries and screening
Yang Xu, College of Life Sciences
Colony and plaque hybridization with probe-III
5. Hybridization: The membrane is
then immersed in a solution
containing a nucleic acid probe,
which is usually radioactive (32Plabeled) and incubated to allow the
probe to hybridize the target gene.
And then, the membrane is washed
to remove unhybridized probe.
6. Auto-radiography: The regions hybridized of the membrane are then
visualized by the exposure to X-ray film.
7. Comparing: By comparing the membrane with the master plate, the
original group of colonies or plaques can be identified.
8. Isolating: The master plate is kept to isolate the corresponding clone
and the recombinant  phage.
9. Re-plating: This group is re-plated at a lower density, and the
hybridization process repeated until every single individual clone is
isolated.
Section I: Gene libraries and screening
Yang Xu, College of Life Sciences
Expression screening-I
Definition: If specific antibodies had been raised to the proteins,
the specific binding to the proteins can be used for screening the
cDNA libraries that express special proteins.
Mechanism:
• Cloning: By cloning cDNAs into the EcoRI site of  gtll, there
is a one in six chance that the cDNA will be in both the correct
orientation and reading frame to be translated into its product.
• Expressing: The EcoRI site is near the C terminus of the lacZ
gene, and the insert must be linked to that of lacZ, in the correct
orientation and frame, for expressing a -gal fusion protein
containing the cDNA gene product.
• Screening: The -gal fusion protein may contain regions of
polypeptide (epitopes) that will be recognized by antibodies
raised to the native protein. These antibodies can therefore be
used for screening the expression library.
Section I: Gene libraries and screening
Yang Xu, College of Life Sciences
Expression screening-II
Procedure:
1. Transferring: It has similarities to the plaque hybridization
protocol (above), though in this case it is the protein encoded by
the cDNA rather than the DNA itself that is transferred on the
membrane.
2. Binding: The membrane is treated to covalently attach the protein,
and immersed in a solution of the antibody. When the antibody has
bound to its epitope, it is detected by other antibodies and/or
chemicals that recognize it.
3. Comparing: By comparing the membrane with the master plate,
the location of the plaque can be narrowed down.
4. Isolating: Repeat cycles of screening are again required to isolate
pure plaques.
Section I: Gene libraries and screening
Yang Xu, College of Life Sciences
Hybrid arrest
Hybrid arrested translation:
cDNAhybridized mRNA proteins
• Hybridization: Individual cDNA
clones or pools of clones can be
hybridized to mRNA preparations. The
hybridized mRNA can not be
translated to proteins, and the detection
of proteins can be used to find which
cDNA can arrest which protein.
• Subdivision: of the pools of cDNA into
smaller numbers and repeating the
experiment should allow the
identification of a single cDNA that
arrests the translation of one protein.
Section I: Gene libraries and screening
mRNA
cDNA
Yang Xu, College of Life Sciences
Hybrid release
Hybrid release translation:
cDNAhybridized mRNAproteins
• Purification: After hybridization, the
hybrids can be purified, e.g. the
[cDNA/mRNA] are attached to
magnetic beads or are precipitable
with an antibody on membrance.
• Release: Then the mRNAs are
released from hybrids (by heat or
denaturant) and translated. This hybrid
release translation identifies which
cDNA clone encode which protein.
Section I: Gene libraries and screening
cDNA
mRNA
mRNA
Yang Xu, College of Life Sciences
That’s all for Section I
Section I: Gene libraries and screening
Yang Xu, College of Life Sciences