DNA ,LIBRARY CONSTUCTION ,BACTERIOPHAGE LAMBDA

1. 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. 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
cDNA libraries
Gene library
(made from genomic DNA)
(made from cDNA- copy of mRNA)
I1 Genomic libraries

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

5. 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= = 1.1 ×103
ln( 1-0.99)
ln[1-(2×104
/4.6×106
)]
Nhuman= = 6.9 ×105
ln(1-0.99)
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.
I1 Genomic libraries

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

7. 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 :
Prokaryotes :extracted DNA directly from cells
remove protein, lipids and other unwanted macro-
molecules by protease digestion and phase extraction.
Eukaryotes :prepare cell nuclei
I1 Genomic libraries

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

9. Sau3A: 5’-/GATC-3’, less selectivity
BamH1: 5’-G/GATCC
Selection of restriction enzyme
1. Ends produced (sticky or blunt) &
The cleaved ends of the vector to be cloned
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.
I1 Genomic libraries

10. Generating A Genomic
Library
 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).
2. λ phage as a higher transformation
efficiency about 1000 times higher
compared to a plasmid.
 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 λ-phage 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. Vectors
According to genome’s size,we can select a
proper vector to construct a library .
Vectors Plasmid phageλ cosmid YAC
insert (kb) 5 23 45 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
I1 Genomic libraries

15. cos cos
Long (left)
arm
short (right)
arm
Exogenous DNA
(~20-23 kb)
λ phage vector in cloning
cos cos
Long (left)
arm
short (right)
arm
Exogenous DNA
(~20-23 kb)

16. λ replacement
vector cloning
2. Packing with a
mixture of the phage
coat proteins and
phage DNA-
processing enzymes
3. Infection and
formation of
plaques
Library constructed
1. Ligation
0.preparation of
arm and genomic
inserts

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

18. 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.
I 2 cDNA libraries

19. 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
I 2 cDNA libraries

20. mRNA isolation
• Most eukaryotic mRNAs are polyadenylated at
their 3’ ends
• oligo (dT) can be bound to the poly(A) tail
and used to recover the mRNA.
AAAAAAAAAAn5’ cap
I 2 cDNA libraries

21. I 2 cDNA libraries

22. 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 mRNA-
ribosome complexes on sucrose gradients
Three methods to isolate mRNA.
I2 cDNA libraries

23. 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
Check the mRNA integrity
I2 cDNA libraries

24. 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)
I2 cDNA libraries

25. 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.
I2 cDNA libraries

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

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

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

29. 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)
I2 cDNA libraries

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

31. 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.
I3 Screening procedures

32. Screening libraries
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
Searching the genes of interest in a DNA library
I3 Screening procedures

33. 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
Containing Nucleic acid probe)
Wash to remove unhybri-
dization probe and visualize
X-ray
film(radio-
actively
labeled )
antibody or
enzyme
(modified
nucleotide
labeled
Line up the hybridizated region or
repeated hybridization
(Alkali treatment)
I3 Screening procedures

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

35. 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
I3 Screening procedures

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

37. 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
I3 Screening procedures

38. 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 .
I3 Screening procedures

39. 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.
I3 Screening procedures

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Vector arm Genomic clone insert Vector arm
Prepare probe from
ends of insert
Re-screen genomic
library
Restriction
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