1. Gene libraries and screening
Genomic libraries
cDNA libraries
Screening procedures

2. Introduction
• The use of genetic information is a
powerful tool that today is becoming more
readily available to scientists.
• In order to use this powerful tool it
necessary to know how to navigate
throughout the entire genome. The human
genome is about 3 x 10E9 bp.
• In humans this project is known as Human
Genome Project.

3. I1 Genomic libraries
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)

4. I1 Genomic libraries
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:
ln (1-P)
N=
ln (1-f)
P: desired probability
f : the fraction of the genome in one insert

5. 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=
Nhuman=
ln( 1-0.99)
ln[1-(2×104/4.6×106)]
ln(1-0.99)
= 1.1 ×103
= 6.9 ×105
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.

6. I1 Genomic libraries
Genomic DNA libraries
eukaryotes
Purify genomic DNA
prokaryotes
Fragment this DNA : physical shearing
and restriction enzyme digestion
Clone the fragments into vectors

7. 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 :
Eukaryotes :prepare cell nuclei
remove protein, lipids and other unwanted macromolecules by protease digestion and phase extraction.
Prokaryotes :extracted DNA directly from cells

8. I1 Genomic libraries
Break DNA into fragments randomly:
Physical shearing :
pipeting, mixing
Restriction enzyme digestion:
partial digestion is preferred
to get a greater lengths of DNA
fragments.

9. I1 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.

10. Generating A Genomic Library
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•
•
•
λ-phage is treated with restriction
enzymes that produce λ arms with
sticky end. These arms contain all the
lytic genetic information that is needed
for replication and produces room for
insertion of new genetic information.
DNA sequence is obtain from the cell of
interest. It is cleaved with restriction
enzymes that produce 20kb fragments
that have complementary sticky ends.
Both are mixed in equal amounts and
are treated with a DNA ligase that
cleaves them together.
Afterward the entire combined sequence
is packed to the phage head.

11. λ-phage as a Vector
•
The genomic library is
generated by using λ-phage for
the following reasons.
1.
A large number of λ phage can be screened
simultaneously (5 x 10E4 phage plagues).
λ phage as a higher transformation efficiency
about 1000 times higher compared to a
plasmid.
2.
•
•
The vector as to maintain its
lytic growth.
Lysogenic pathway and other
viral genes that are not
important are replaced with the
DNA to be cloned.

12. λ-phage as a Vector (Cont.)
•
•
•
An infected E.Coli will produce
what are know as concatomers
(which is the viral genome) on either
site of the concatomers there is a site
called COS Site.
Two proteins recognize this site A
protein and Nu protein, which will
lead to the insertion of the λ DNA
into the phage head. The
chromosomal DNA that lacks the
COS sites will not enter the phage
head. Once the genetic information
is inserted the tail will assemble.
A 50kb can be inserted into the
phage.

13. Packaging of the Recombinant DNA
•
•
•
•
•
•
•
To prepare the phage an E.coli cell is infected with a mutant λ-phage
that as a defective “A-protein” (which is one of two genes that are
responsible for packaging genetic information).
Therefore the E.Coli accumulates empty heads and also preassembled
tails.
Once enough heads and tails are assembled we lysate the E.Coli cells.
To the mixture of heads and tail we add isolated A protein (obtained
from E.Coli infected with λ-phage).
In the next step we add the recombinant DNA that has the λ-phage
genetic information (which also includes COS sites).
At this point we have a mixture containing mutant λ-phage heads and
tails. There is isolated A protein and recombinant DNA containing λphage genetic information with COS sites.
Therefore we have all the components necessary to package the
recombinant DNA into the λ-phage head. Once the information is
inserted the tail assembles and we have an infectious phage that
contains the recombinant DNA sequence.

14. I1 Genomic libraries
Vectors
According to genome’s size,we can select a
proper vector to construct a library .
Vectors
Plasmid
insert (kb)
5
phageλ
23
cosmid
45
YAC
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

15. 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)

16. 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

17. Gene libraries and screening
I
cDNA libraries
mRNA isolation, purification
Check theRNA integrity
Fractionate and enrich mRNA
Synthesis of cDNA
Treatment of cDNA ends
Ligation to vector

18. I 2 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.

19. I 2 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)

20. I 2 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.

21. I 2 cDNA libraries

22. I2 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

23. I2 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

24. I2 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)

25. I2 cDNA libraries
Synthesis of cDNA :
First stand synthesis: materials as
reverse transcriptase ,primer( oligo(dT) or
hexanucleotides) and dNTPs
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.

26. I2 cDNA libraries
5’
5’
3’
5’
3’-CCCCCCC
5’-pGGGG-OH
3’-CCCCCCC
5’-pGGGG
3’-CCCCCCC
mRNA
Reverse transcriptase
Four dNTPs
mRNA
AAAAA-3’
HO-TTTTTP-5’
cDNA
AAAAA-3’
TTTTTP-5’
cDNA
AAAAA-3’
TTTTTP-5’
Terminal transferase
dCTP
mRNA
Alkali (hydrolyaes RNA)
Purify DNA oligo(dG)
TTTTTP-5’
Klenow polymerase or reverse
Transcriotase Four dNTPs
-3’
TTTTTP-5’
Duplex cDNA
cDNA
The first strand synthesis

27. Duplex cDNA
5’-pGGGG
3’-CCCCCCC
-3’
TTTTTp-5’
Single strand-specific nuclease
5’-pGGGG
3’-CCC
-3’
TTTTTp-5’
Klenow polymerase
treat with E.coRI methylase
5’-pGGGG
3’-CCCC
Add E.colRI linkers
using T4 DNA ligase
HO-CCGAATTCGGGGGG
3’-GGCTTAAGCCCCCC
-3’
TTTTTp-5’
HO-CCG/AATTCGG-3’
3’-GGCTTAA/GCC-OH
CCGAATTCGG-3’
TTTTTGGCTTAAGCC-OH
E.colRI digestion
5’-pAATTCGGGGGG
3’-CCCCCCC
CCG-3’
TTTTTGGCTTAAp-5’
Ligate to vector and transfom
Second strand synthesis

28. I2 cDNA libraries
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)
Tailing with terminal transferase or
using adaptor molecules
Restriction enzyme digestion (E.coRI )

29. I2 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)

30. Gene libraries and screening
Screening procedures
Screening
Colony and plaque hybridization
Expression screening
Hybrid arrest and release
Chromosome walking (repeat screening)

31. I3 Screening procedures
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.

32. I3 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

33. I3 Screening procedures
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

34. I3 Screening procedures
Expression screening
Identify the protein product of an
interested gene
1. Protein activity
2. Western blotting using a specific
antibody

35. I3 Screening procedures
Expression screening
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

36. I3 Screening procedures
Expression screening
Antibodies can be used to screen the
expression library.
The procedure
‘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

37. I3 Screening procedures
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

38. I3 Screening procedures
I3-5 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 .

39. I3 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.

40. 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

41. Thanks